Automated method and device for identifying and quantifying platelets and for determining platelet activation state using whole blood samples

ABSTRACT

The present invention provides a highly sensitive and accurate method and system for the discrimination and quantification of platelets in a whole blood sample using automated hematology instruments. The method and system of the invention provide the accurate measurements of platelet dry mass and platelet component concentration in both normal blood samples and in abnormal blood samples, such as those from thrombocytopenic patients. The determination of platelet dry mass and platelet component concentration can serve to assess the activation state of platelets since activated platelets possess measurably lower component concentrations and refractive indices than do unactivated platelets. The method and system of the invention also allows the clinician or skilled practitioner to determine the age of a blood sample on the basis of the measured parameter of platelet component concentration.

This application is a continuation-in-part of application Ser. No.08/581,293 filed on Dec. 28, 1995, now abandoned.

FIELD OF THE INVENTION

The present invention relates to improved methods of platelet analysisand quantification using automated hematology systems. In particular,the invention relates to a high-gain method that can be used for bothnormal and abnormal blood samples to determine among other parameters,platelet count, platelet volume, platelet component concentration ordensity, and platelet dry mass with a higher degree of accuracy andprecision compared with other methods.

BACKGROUND OF THE INVENTION

Although semi- and fully-automated analyzer systems are now routinelyused to determine blood platelet counts, it is recognized in the artthat current automated platelet determination and quantification methodsare still hampered by problems of inaccuracy, lack of precision, andlack of reproducibility. This is particularly evident in the analysis ofabnormal blood samples, such as those obtained from individuals orpatients afflicted with a number of blood dyscrasias and thromboticdisorders such as thrombocytopenia (a decrease in the number of bloodplatelets), thrombocytosis, and the like. Several reasons for thedifficulties and challenges in controlling accuracy of platelet countsmay be attributed to 1) the large dynamic range of the platelet countand size for patients; 2) the small size of platelets; 3) the presenceof interfering particles of platelet size in the samples undergoinganalysis; and 4) the behavior of platelets upon in vitro aging.

Platelet analysis and quantification can be especially difficult in thecase of thrombocytopenic individuals who have reduced or low numbers ofplatelets in their blood samples. This condition frequently results fromtreatments and therapies commonly used for cancer patients who havedecreased thrombotic tendencies. In addition, individuals afflicted withcertain immunologic diseases, particularly autoimmune disease, such asidiopathic thrombocytopenic purpura (ITP), often suffer platelet damageand destruction leading to decreased platelet numbers. Further,individuals suffering from aplastic anemia(s) also have reduced numbersof blood platelets. For example, in samples from thrombocytopenicindividuals, platelet numbers are often less than 50,000/μl, comparedwith platelet numbers in the normal range which are on the order ofabout 150,000-400,000/μl (J. C. Dacie and S. M. Lewis, 1984, PracticalHaematology, 6th Edition, Churchill Livingstone, London).

Indeed, the accurate enumeration of platelets became even more importantwith the advent of widely-available platelet replacement therapies forthrombocytopenic patients. In addition, the development and use of avariety of more sophisticated studies of platelet function, e.g.,platelet activation and/or adhesiveness determinations, require accurateand precise platelet counts as an integral part of the laboratoryhematology test. Also, the quality assurance of platelet packs preparedfor transfusion requires that accurate platelet counting procedures beavailable and in place (R. K. Wertz and J. A. Koepke, 1977, Am. J. Clin.Path., 68(1):195-201).

Needed in the art are more accurate, precise, and sensitive methods forthe detection, discrimination, quantification, and characterization ofthe parameters of platelets for both normal and abnormal blood samples.In addition, accurate and precise platelet analysis methods that can beperformed using whole blood samples obviate the initial preparation ofplatelet-rich plasma by differential centrifugation or sedimentationtechniques that is required by some methods. Such whole-blood plateletanalysis methods may be useful for diagnosing unsuspected plateletabnormalities, as well as for monitoring platelet counts and parametersof normal individuals and of patients at the onset of a disorder andduring the course of treatment or progression of disease. In addition,methods for use on automated systems that can improve the signalresolution and the discrimination of platelets, especially in cases oflow platelet counts, are needed in the art. The present invention asdescribed provides such advantages and improvements to the art.

Two fully automated platelet counting and sizing methods are currentlyknown and used by those in the art. One is the aperture impedancemethod. Whole-blood platelets in aqueous suspension are detected as theypass through a narrow aperture located between two electrodes, therebyincreasing the electrical impedance in the aperture relative to that ofthe suspending medium in rough proportion to platelet volume. Thus, theplatelet pulses provide platelet count and platelet volume. Plateletsare distinguished from red blood cells in the aperture impedance methodon the basis of their size, since platelets as a group are smaller thanred blood cells as a group. In some applications of this method,platelet count and size determinations are refined by mathematicalanalysis of the shape of platelet size distributions. Thesedistributions are fitted to log-normal curves and the parameters of thefitted curves provide platelet count and size. Although the intent ofthis treatment is to exclude particle debris and small red blood cellswhose presence distorts the log-normal platelet volume distribution,such contaminating particles and cells are not always excluded.

A second fully automated method is the laser light scatter method. Inthis method, whole-blood platelets in aqueous suspension are detected asthey intercept a laser beam, thus causing the incident light to scatterat characteristic angles into paths in which optical detectors areplaced. The platelet signal pulses provide volume information as well ascounts, since platelet volume is considered to be proportional toscattering intensity. Examples of automated flow cytometry instrumentswhich have been designed and are employed to carry out such lightscattering methods are the H•™System instruments (commercially availableunder the trade designation TECHNICON H•™Systems, e.g., H•™1, H•™2,H•™3, and the like, and sold by the assignee hereof) and the ORTHO ELT-8(Ortho Diagnostics).

In the ORTHO ELT-8 system, platelets are distinguished from red bloodcells simply by differences in scattering intensity over a single coneangle. In the TECHNICON H•™Systems, platelets are also sized on thebasis of scattering intensity over a single cone angle; however, theyare distinguished from red blood cells on the basis of theircharacteristic scattering intensities into a pair of suitably chosendetectors. Although the platelet scattering intensity distribution islog-normal, the second laser light scattering method does not refinecounts or sizing by fitting the data obtained using log-normal curves.Particle debris in this method is comprised of signals whose scatteringintensities fall below a selected threshold.

As mentioned above, the aperture impedance method distinguishesplatelets from red blood cells and particle debris on the basis ofparticle size, as well as on the basis of the log-normal distribution ofplatelet sizes. In cases where platelets and other particles are ofoverlapping size, these distinctions blur, and the best that the methodcan do is to recognize this failure. Moreover, the light scatteringmethod distinguishes platelets from red blood cells based ontwo-dimensional boundaries, which may be crossed when red blood cellsbecome small or if they fragment, thus also blurring the distinctionbetween the disparate cell populations.

A third, semi-automated method for platelet discrimination involves acombination of laser light scattering and fluorescence to distinguishplatelets from red blood cells and particle debris. Whole-bloodplatelets in aqueous suspension are labelled with plateletspecific-antibodies, such as CD42A. The antibodies, in turn, are boundto fluorophores such as fluorescein isothiocyanate (FITC). The labelledplatelets scatter incident light and fluoresce as they pass through afluorescence flow cytometer, such as a Becton Dickinson FACScan (BectonDickinson). The platelets and platelet-sized particles are distinguishedfrom red blood cells on the basis of two dimensional scattering patterns(forward scatter and side scatter). These "gated" cells are furtherclassified on the basis of fluorescence intensity; with only plateletsdisplaying significant fluorescence (W. Groner et al., 1994, Blood, No.10 Supplement, 687a; R. Dickerhof and A. von Ruecker, 1995, Clin. Lab.Hematol., 17:163-172).

Although the last two above-described methods allow the discriminationof platelets from other blood cell types and from debris, they do notprovide absolute platelet counts. Furthermore, they do not determineplatelet size, since there is no simple way to calibrate the methods andthe systems performing the methods for this purpose. In addition, thelabelling technique is labor-intensive and relatively expensive.

In addition to discriminating and quantifying platelets in bloodsamples, simple, inexpensive, accurate and reproducible methods fordetermining platelet activation (or activation state) are needed in theart. The activation state of platelets is an important parameter ofplatelet function as described below.

Platelet activation is a fundamental functional property of platelets,since activated platelets play an integral role in hemostasis andthrombos. When vascular injury occurs, subendothelial surfaces areexposed at the site of injury, which results in the adhesion ofactivated platelets to the subendothelial surface. This is followed byplatelet granule release, platelet aggregation and thrombus formation.Thrombi are composed of fibrin, platelet aggregates and red blood cells.

Activated platelets are distinct from resting platelets in that theformer express surface glycoproteins associated with the adhesionprocess. Also, activated platelets release granular components andundergo such processes as the disk-to-sphere shape change andaggregation. Swelling is also associated with the shape change.

Thrombosis is part of the normal response to vascular injury. However,increased thrombotic activity also occurs, with negative effects, inconditions such as peripheral vascular disease (D. V. Devine et al.,1993, Arteriosclerosis and Thrombosis, 13:857-62), cardiac ischemia (D.McTavish et al., 1990, Drugs, 40:238; G. DiMinno et al., 1985, J. Clin.Invest., 75:328), diabetes mellitus (D. Tschoepe et al., 1991, Seminarsin Thrombosis and Hemostasis, 17:433-438) and angina (R. C. Becker etal., 1994, Coronary Artery Dis., 5:339). It is also known thatblood-banked platelets in concentrates become activated during storageand, as a result, lose some of their potency (H. M. Rinder and E. L.Snyder, 1992, Blood Cells, 18:445). In addition, hemodialysis andsurgical procedures involving extracorporeal circulation of blood areknown to cause platelet activation (e.g., J. C. Reverter et al., 1994,J. Lab. Clin. Med., 124:79; Y. T. Wachtfogel et al., 1993, J. Thoracicand Cardiovascular Surg., 106:1-10; R. E. Scharf et al., 1992,Arteriosclerosis and Thrombosis, 12:1475-1487). Accordingly, the abilityto identify and monitor the activation state of platelets ex vivoprovides an advantageous and useful screening technique afforded by thepresent invention.

Platelet activation has been studied using fluorescence flow cytometry(e.g., S. J. Shattil et al., 1987, Blood, 70:307; C. S. Abrams et al.,1990, Blood, 75:128; L. Corash, 1990, Blood Cells, 16:97-108). Usingfluorescence technology, platelets are marked withfluorescence-conjugated antibodies specific to glycoproteins that areexpressed, or that undergo conformational changes, on the plateletsurface as a result of platelet activation. The number offluorescence-positive events counted on a flow cytometer represents thenumber of activated platelets; the fluorescence intensity per eventrepresents the number of marked sites per cell surface. Although thistechnique is specific and sensitive, it is also disadvantageous inseveral ways, namely, it is expensive; sample preparation istime-consuming; and data analysis is not automated. Further, no standardmethod has been established for setting fluorescence-positivethresholds, partly because of the arbitrary nature of the thresholdposition and partly because of differences in experimental design.

Platelet activation has also been studied by density-gradient analysis(B. van Oost et al., 1983, Blood, 62:433-38). The density of plateletsdrops as they are activated, primarily due to swelling and secondarilydue to the release of alpha- and dense-granules (S. Holme et al., 1981,J. Lab. Clin. Med., 97:610-22; S. Holme et al., 1988, J. Lab. Clin.Med., 112:223-231) which are denser than the platelet cytoplasm.Consequently, activated platelet samples have higher percentages oflow-density platelets in density-gradient separations than donon-activated samples. The density-gradient separation technique is timeconsuming and requires a skilled technologist. Further, a cell counteris required to determine the number of platelets in each of thedensity-gradient fractions.

Accordingly, the present invention which offers a novel, inexpensive andsensitive absorption light scattering technique for the determination ofplatelet activation provides an advancement and advantage to the art.The present method of determining platelet activation is automated andthus is efficient and time-saving for clinical use.

SUMMARY OF THE INVENTION

The present invention provides a sensitive, accurate, and precise methodfor the quantification and analysis of platelets in whole blood samplesand is particularly useful for analyzing blood samples from individualswho have abnormal blood conditions which adversely affect the numbersand/or discrimination of blood platelets. The invention also affords aquick, simple, and inexpensive method, system, and apparatus forplatelet discrimination and analysis.

It is an object of the present invention to provide an improved methodof platelet analysis using automated hematology systems for gatheringplatelet data, including platelet count, size, component concentrationand dry mass.

It is another object of the invention to provide a high-gain orhigh-amplification method and apparatus for platelet counting accuracythat offers more platelet analysis data and provides results moreinexpensively and easily than methods which employ fluorescenceintensity, scattering intensity, and aperture impedance fordistinguishing non-platelets from platelets in a blood sample.

Yet another object of the invention is to provide a method of plateletcounting, signal resolution, and discrimination that is sensitive andaccurate for blood samples having platelet counts of from about 1,000 toless than about 50,000 platelets per microliter. In particular, theinvention provides improved platelet count accuracy for thrombocytopenicsamples.

Still another object of the invention is to yield cytogram results thatprovide well delineated and detailed depictions of particle-typedistribution in the platelet-size region.

Another object of the invention is to provide a means to modify andimprove current automated hematology analyzers by the addition of atleast two channels to perform the improved method of platelet countingand analysis. The modification or improvement adds a low scatteringangle/high-gain amplification channel and a high scatteringangle/high-gain amplification channel to current systems plus MieScattering Theory-based analysis of signals to achieve the performanceof the method and apparatus of the invention.

Another object of the invention is to allow the identification andquantification of microcytic red cells and red blood cell fragments.

Yet another object of the invention is to provide a determination of theextent of platelet activation by the measurement of platelet componentconcentration (MPC). In accordance with the invention, MPC values arecorrelated with platelet activation state.

A further object of the invention is to provide a determination ofplatelet dry mass which is a predictor of platelet activatability.

Another object of the invention is to provide a determination ofplatelet component concentration which allows the measurement of invitro blood sample age.

Further objects and advantages afforded by the invention will beapparent from the detailed description hereinbelow.

DESCRIPTION OF THE DRAWINGS

The appended drawings of the figures are presented to further describethe invention and to assist in its understanding through clarificationof its various aspects. The scatter/scatter cytograms as describedhereinbelow were obtained when the present apparatus and method of theinvention were employed in the determination and analysis of plateletsusing an electro-optical detection system of an automated hematologyanalyzer in accordance with the invention. In the figures presentedhereinbelow, RBC=red blood cell count in 10⁶ /μl; PLT=platelet count in10³ /μl; MPV=mean platelet volume in femtoliters (fl); MP+C isequivalent to MPC=mean platelet component concentration in g/dl; andMP+M is equivalent to MPM=mean platelet dry mass in picograms (pg).

FIG. 1 depicts a platelet volume (V) versus refractive index (n) (i.e.,V/n) map on a scatter/scatter cytogram. Each line shown in the cytogramrepresents a particular particle type as determined in the analysis andas identified in the description of FIG. 3.

FIGS. 2A, 2B, and 2C show scatter/scatter cytograms of n-pentane,n-hexane, and n-heptane oil droplets, respectively. Each line shown inthe cytogram represents a particular particle type as determined in theanalysis and as identified in the description of FIG. 3.

FIG. 3 demonstrates the locations of various particles, i.e., red bloodcells, large platelets, red blood cell ghosts, platelets, red blood cellfragments, and origin debris in a particle-type mapping scatter/scattercytogram. This description also applies to the cytograms shown in FIGS.4, 5, 6, 8, and 13.

FIG. 4 depicts a representative cytogram and histograms showing a normalsample result output using the platelet determination method and systemof the invention.

FIG. 5A depicts a representative cytogram resulting from the analysis ofplatelets in K₃ EDTA-anticoagulated blood samples suspended in red bloodcell diluent (e.g., TECHNICON RBC Diluent). FIG. 5B depicts arepresentative cytogram resulting from the analysis of platelets inK,EDTA-anticoagulated blood samples suspended in isotonic phosphatebuffered saline (PBS). It is to be understood that samples are analyzedat room temperature, even if they have not been stored at roomtemperature.

FIG. 6A depicts a representative cytogram resulting from the analysis ofACD-anticoagulated blood samples suspended in red blood cell diluent(e.g., TECHNICON RBC Diluent). FIG. 6B depicts a representative cytogramresulting from the analysis of ACD-anticoagulated blood samplessuspended in isotonic phosphate buffered saline (PBS).

FIG. 7A is a representative graph showing mean platelet dry mass (MPM)versus time for normal blood donor samples. MPM was determined for 24hour old normal samples versus 1 hour old normal samples stored at roomtemperature.

FIG. 7B shows a representative graph showing mean platelet dry massversus time for abnormal blood donor samples. MPM was determined for 28hour old abnormal samples versus 4 hour old abnormal samples stored atroom temperature.

FIGS. 8A, 8B, and 8C depict platelet volume (MPV) histograms for anabnormal sample generated by several automated methods, i.e., the PLT1system of the invention (FIG. 8A), the TECHNICON H•™2 System (FIG. 8B),and the Coulter STKS System (FIG. 8C).

FIGS. 9A-9D depict mean platelet volume (MPV) versus abnormal platelet(PCT) count determinations (PLT count<20,000/μl) obtained using eitherthe TECHNICON H•™2 System or the Coulter STKS System. FIG. 9A shows theresults obtained from the TECHNICON H•™2 System using 4 hour oldabnormal blood samples. In FIG. 9A, r=0.59; Syx=0.88 (Please define rand syx); slope=0.14; and intercept=4.5. FIG. 9B shows the resultsobtained from the TECHNICON H•™2 System using 28 hour old abnormal bloodsamples. In FIG. 9B, r=0.72; Syx=0.54; slope=0.13; and intercept=3.29.FIG. 9C shows the results obtained from the Coulter STKS System using 4hour old abnormal blood samples. In FIG. 9C, r=0.13; Syx=0.95;slope=0.02; and intercept=8.06. FIG. 9D shows the results obtained fromthe Coulter STKS System using 28 hour old abnormal blood samples. InFIG. 9D, r=0.66; Syx=1.09; slope=0.18; and intercept=6.31.

FIGS. 10A and 10B depict the mean platelet volume (MPV) versus platelet(PCT) count determination for abnormal thrombocytopenic blood samples(PLT count: <20,000/μl) obtained using the novel PLT1 System and methodof the invention. FIG. 10A shows the results obtained from the PLT1System using 4 hour old abnormal blood samples. In FIG. 10A, r=0.32;Syx=0.89; slope=-0.06; and intercept=10.02. FIG. 10B shows the resultsobtained from the PLT1 System using 28 hour old abnormal blood samples.In FIG. 10B, r=0.32; Syx=1; slope=-0.07; and intercept=11.79.

FIGS. 11A, 11B, and 11C depict mean platelet volume (MPV) versusplatelet (PCT) count determinations performed on normal blood samplesobtained using different automated methods, namely, the TECHNICON H•™2System, the Coulter STKS System, and the novel PLT1 System of theinvention. FIG. 11A shows the results obtained from the TECHNICON H•™2System using 1 hour old normal blood samples; FIG. 11B shows the resultsobtained from the Coulter STKS System using 1 hour old normal bloodsamples; and FIG. 11C shows the results obtained from the PLT1 System ofthe invention using 1 hour old normal blood samples.

FIGS. 12A and 12B show the mean platelet dry mass (MPM) and meanplatelet component concentration (MPC) accuracy data for abnormalsamples using the PLT1 method and system. FIG. 12A depicts MPM accuracydata for 28 hour old abnormal samples versus 4 hour old abnormal samplesstored and analyzed at room temperature. In FIG. 12A, r=0.75; Syx=0.13;slope=0.74; intercept=0.49; the 4 hour mean value is 2.04; and the 28hour mean value is 1.99. FIG. 12B depicts MPC accuracy data for 28 hourold abnormal samples versus 4 hour old abnormal samples at roomtemperature. In FIG. 12B, r=0.35; Syx=1.3; slope=0.3; intercept=12; the4 hour mean value is 22.1; and the 28 hour mean value is 18.7.

FIGS. 13A, 13B and 13C depict the platelet volume (MPV) histograms,which correspond to the results presented in Table 8 for abnormalthrombocytopenic sample #70. FIG. 13A represents the histogram resultsobtained using the PLT1 method of analysis of the invention; FIG. 13Brepresents the histogram results obtained using the TECHNICON H•™2System of analysis; and FIG. 13C represents the histogram resultsobtained using the Coulter STKS System method of analysis.

FIGS. 14A to 14D show a comparison between the novel light scatteringmethod in accordance with the invention and a fluorescence method fordetermining platelet activation. FIGS. 14A-D demonstrate that the novellight scattering method (FIGS. 14C and 14D) of the invention and thefluorescence method (FIGS. 14A and 14B) track thrombin dose-relatedplatelet activation in a similar manner for both sodium citrate-treatedand EDTA-treated blood samples. The dose response curves for the methodof the invention are presented in standard format with MPC increasing

(FIG. 14C) and in "upside down" format with MPC decreasing (FIG. 14D).Both the upside down and the standard format curves display identicalresults, but the former orientation permits a more direct visualcomparison of the results between the two methods (i.e., FIG. 14Bdirectly compared with FIG. 14D).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a sensitive and accurate method ofplatelet quantification and characterization for use on automatedhematology analyzer systems. The invention further provides an apparatusfor performing the platelet quantification and characterization methodsas described. As used herein, platelet is frequently abbreviated "PLT".Other abbreviations frequently used herein are the following: RBC is redblood cell; MPM is mean platelet dry mass; MPV is mean platelet volume;MPC is mean platelet component concentration; TCP is thrombocytopenic orthrombocytopenia, and TCPS is the designation used for an abnormal bloodsample obtained from a patient with thrombocytopenia; MCV is mean cellvolume; MCHC is mean cell hemoglobin concentration; and HCT ishematocrit.

Current types of automated hematology analyzer systems suitable for usewith the invention are exemplified by the H•™Systems commerciallyavailable under the trade designation TECHNICON H•™1, H•™2, H•™3, andthe like, sold by the assignee hereof. In such systems, platelet (orparticle) detection is made electro-optically by measuring light scatterand electrically by measuring electrical impedance. For those skilled inthe art, the operating principles of the TECHNICON H•™ automatedanalyzer systems are set forth herein with respect to red blood cell andplatelet analysis in order to clearly describe the changes made to suchsystems to produce the invention. In these systems, red blood cells andplatelets are analyzed together in a single optical measurement channel,which includes a Helium-Neon laser light source, a flowcell, and twooptical detectors. As part of its normal operating procedure, theautomated system suspends two microliters (2 μl) of whole blood in 1.25ml of TECHNICON H•™Systems RBC Diluent, a reagent solution whichisovolumetrically spheres red blood cells so that they may be properlyanalyzed using Mie Scattering Theory, as explained herein. Red bloodcell sphering reagents and diluents suitable for use in the plateletanalysis method and system of the invention are described in U.S. Pat.Nos. 5,284,771, 5,360,739 and 5,411,891 to S. S. Fan et al.; in U.S.Pat. Nos. 5,350,695 and 5,438,003 to G. Colella et al.; and in U.S. Pat.Nos. 4,575,490 and 4,412,004 to Kim and Ornstein.

A stream of approximately 10 μl of this suspension is then sheathed in areagent fluid of matching refractive index, i.e., the TECHNICONH•™Systems RBC/Basophil surfactant sheath. The RBC/Basophil sheath is a"passive" reagent which does not interact with blood cells directly, butinstead surrounds and centers the stream in the flowcell. It alsoprovides a procession of single particles for analysis. For example, theRBC/Basophil surfactant sheath reagent composition comprises inorganicsalt, such as sodium chloride, 7.7 g/l; sodium phosphate, dibasic, 2.4g/l; sodium phosphate, monobasic, 0.3 g/l; the polyethoxylate nonionic,nonhemolytic surfactant Pluronic P-105, 1.0 g/l; an antioxidant reagent,such as 3,3' thiodipropionic acid, 0.10 g/l; and an antimicrobialreagent, such as Proclin 150, 0.40 g/l, at a pH of about 7.0-7.5 and anosmolality of about 285-305 mOsmol/kg.

The red blood cells and platelets in the suspension scatter some of theincident laser light as the sheathed stream of cells passes through theflow cell. The two detectors sense the light scattered at particularangular intervals relative to the axis of incidence. The detectorsignals are amplified so that, for normal samples, the average signalsproduced by red blood cells are in the middle of the range of signalamplitudes associated with red blood cells. In accordance with theinvention, the light scattering intensity is measured over two coneangle intervals in two optical channels at increased first and secondoptical channel signal gains to produce two scattering intensitymeasurements sufficient to resolve the platelets from the non-plateletsin the sample. The first optical channel signal value derives from anincrease in the gain of the high angle detector and the second opticalchannel signal value derives from an increase in the gain of the lowangle detector, thereby resulting in a novel high gain version of thelow and high angle outputs. Also in accordance with the invention, thesystem displays the signals due to degree scatter of approximately 5 to20 degrees, more preferably 7 to 15 degrees, and most preferably, 5 to15 degrees along the X-axis of a scatter/scatter cytogram. In addition,the system displays signals due to degree scatter of approximately 1 to7 degrees, more preferably 1 to 5 degrees, and most preferably, 2 to 3degrees along the Y-axis of the scatter/scatter cytogram. These signalsare used to determine red blood cell parameters such as volume andhemoglobin concentration, as described further herein.

Red blood cells are normally biconcave disks, whose scatteringproperties are sensitive to orientation as they traverse the flow cellof the above-described optical system. In order to eliminate the effectsof particle orientation on signal intensity, red blood cells are spheredin the TECHNICON H•™System RBC Diluent (e.g., U.S. Pat. Nos. 4,575,490and 4,412,004 to Kim and Ornstein). Further, Mie Scattering Theory,which provides angular scattering intensity profiles for spheredparticles (such as sphered red cells), predicts that scatteringintensity is sensitive to refractive index as well as to cell volume.Thus, two particles having equal volume, but having different refractiveindices will have different scattering profiles. Since red blood cellrefractive index depends linearly upon cellular hemoglobin concentration(R. Barer and S. Joseph, 1954, Ouarterly Journal of MicroscopicalScience, 95:399-423), which varies from cell-to-cell, measurements ofscattering intensity over a single cone angle interval is not likely touniquely determine cell volume, even for sphered red blood cells.Therefore, to uniquely determine the volume of a red blood cell, it isnecessary to measure its scattering intensity over at least two separatecone angle intervals. Cellular hemoglobin concentration is determined asa byproduct of the two-angle measurement.

Algorithms using Mie Theory provide the angular scattering patternsassociated with particles of given volume and refractive index. Over therange of red blood cell sizes and concentrations of interest (i.e.,about 30-180 fl and about 19-49 g/dl, respectively), it has beendetermined that a one-to-one correspondence exists between 1) the pairof scatteriing intensities at 2-3 degrees and 5-15 degrees, and 2) thevolume and concentration of red blood cells (U.S. Pat. No. 4,735,504 toTycko). For the TECHNICON H•™Systems, the set of correspondences istabulated in the form of a two dimensional matrix. The indices of thematrix are comprised of the X- and Y-channel signal values and theentries of the matrix are the associated volumes and concentrations.Electromagnetic scattering theory for spherical particles (i.e., MieTheory) has been described in detail, for example, by M. Kerker, 1969,In: The Scattering of Light, Academic Press.

Before automatically applying the Mie table, the system counts as bloodcells all particles that exceed a pre-determined signal threshold. Itthen uses the two above-mentioned cone-angle measurements to designatethem as either red blood cells or platelets. The two particle typesoccupy distinct regions of the volume/refractive index space, with redblood cells being much larger and having significantly higher refractiveindices. However, the signal gains currently used in these systemsprovide poor discrimination of platelets from non-platelets, such as redblood cell ghosts, red cell fragments, and cellular debris. In addition,the current aperture impedance method, which relies on differences insize to distinguish platelets from other particles, does not adequatelydiscriminate platelets from red cell fragments or debris, which havevolumes similar to the volume of platelets.

By contrast, current electro-optical instruments rely on singleangle-interval measurements to determine platelet volume. In the currentH•™Systems, platelet volume is considered to be proportional to the 5-15degree scattering intensity. In fact, as will be described herein, thisintensity decreases with in vitro sample age due to platelet swelling(see Example 1). As a result, the reported mean platelet volume falls asthe true mean platelet volume rises. Also, the single-angle method canreport different volumes for platelets that actually have the samevolumes, but different densities (i.e., refractive indices). Further,both electro-optical and aperture impedance instruments oftenunder-report MPV values for thrombocytopenic samples, because low-signaldebris typically comprises a significant fraction of the total particlecount in these abnormal samples.

As part of the efforts of the present inventors to achieve moresensitive, accurate, and precise platelet analyses for performance onautomated analyzer systems, such as, for example, the TECHNICON H•™analyzer systems, a test station using the TECHNICON H•™ was adapted andconfigured as described herein for gathering platelet data using themethod and system described by the invention, which comprises increasedamplification of both the X- and the Y-red blood cell optical channelsignals (amplified signal gains).

The newly-configured automated system for performing the plateletdiscrimination analyses of the invention is referred to herein as thePLT1 system and method of the invention. For the accurate analysis ofplatelets, Mie Scattering Theory was used to discover the appropriateincreased amplification factors as described herein. First, volume andrefractive index ranges were selected for Mie analysis. The volume rangeselected was about 1 to 60 fl (fl=femtoliters=10₋₁₅ liters), andpreferably 1 to 30 fl. This range was selected to cover platelet sizesfor all normal and most abnormal samples (J. M. Paulus, 1975, Blood,46(3):321-336). The refractive index range selected was about 1.340 to1.400, preferably 1.350-1.400. The lower limit is based on theobservation that platelets have higher refractive indices than theirplasma media (otherwise they would be invisible) and the refractiveindex of plasma is typically greater than about 1.345, as determined byrefractometry. The upper limit is based on the observation that mostplatelets are less dense (and thus, have lower refractive indices) thanred blood cells, whose refractive indices rarely drop below 1.390(cellular hemoglobin concentration of 23 g/dl). The limit was preferablyextended to about 1.400 to account for the small fraction of plateletsthat are as dense as some red blood cells. Based on these volume andrefractive index ranges and Mie Theory, the standard H•™System X-channelsignals were increased or amplified about 8 to 15-fold, preferably about12-fold, and the Y-signals were increased or amplified about 20 to35-fold, preferably about 30-fold in accordance with the invention.

At these signal gain amplifications, light scattering intensities at,for example, 2 to 3 degrees and at, for example, 5 to 15 degrees wereadequate to resolve platelets from interfering substances such as celldebris, red blood cell fragments, and red blood cell ghosts. Also, redblood cells were distinctly discerned in the method, because theirsignals appeared in saturation channels X=99, Y=99. Therefore, thecombined scattering measurements described for the invention providemore accurate platelet counts than do current automated methods,particularly for thrombocytopenic samples, in which the fraction ofinterferences typically increases. Further, the scatter/scattercytograms generated in accordance with the invention permit a visualassessment of the number, average size, and average refractive index ofthe platelets contained in a sample. The cytograms also provide a visualassessment of the numbers and types of other platelet-sized particlesthat may be present in the sample.

In addition, in another aspect of the invention, the two anglescattering measurements that are unique to the invention distinguishplatelets that have released their granules (i.e., activated platelets)from those that have not, since platelets in the activated form havelower refractive indices than those in the unactivated form. Currentautomated instruments do not make this distinction.

Moreover, the high amplification allows the platelet determinationsystem of the invention to perform Mie Theory analysis on the platelets,thereby providing platelet volume and refractive index information basedon theory rather than empirical observation. In the PTL1 test stationsystem, the five parameters measured in accordance with the inventionwere the red blood cell count (RBC count, 10⁶ /μl), the platelet count(PLT count, 10³ /μl), the mean platelet volume (MPV, fl), the meanplatelet dry mass (MPM, pg), and the mean platelet componentconcentration or density (MPC, g/dl).

It is to be understood that prior to the newly-discovered method ofplatelet determination and its testing in the automated test system asdescribed herein, the analytical, measured parameters of MPM and MPCwere not previously available in automated platelet analysis andmeasurement methods and systems known and used in the art. Thus, themethod and system of the invention provides an accurate and completeplatelet analysis of a sample, including platelet count, mean plateletvolume, mean platelet dry mass, and mean platelet componentconcentration. In addition, the invention also provides the sample's redblood cell count at the same time that it produces the complete plateletanalysis as described further hereinbelow.

The high-amplification method of the invention was compared with othermethods with respect to platelet counting and sizing accuracy,sensitivity, and qualitative and quantitative reproducibility. The othercomparative methods included scattering intensity (e.g., the TECHNICONH•™2 System, Bayer Corporation, Tarrytown, N.Y.), aperture impedance(e.g., the Coulter STKS Model system, Coulter Electronics, Dade, Fla.),phase contrast microscopy, and histological blood smear estimates.

The High-Amplification System of PLT1

In accordance with an aspect of the invention, the plateletdiscrimination and quantification method and system of the inventionemploys a newly-determined high-amplification method. The measurementsuniquely developed for the present invention were made on a modifiedTECHNICON H•™1 System, which was named the PLT1 model or paradigmsystem/method, due to its ability to perform unique measuring andquantification parameters on platelets and its distinction from currentautomated systems and devices in the art. For red blood cell analysis,the X-channel signal of the PLT1 system, was amplified about 12-foldfrom the nominal value and the Y-channel signal was amplified about30-fold.

Thus, as described hereinabove and in accordance with the invention, aMie Scattering Theory table was prepared for spheres of about 1 to 30 fland refractive indices of about 1.350 to 1.400. The table is of the sameformat as that used for red blood cell analysis. The volume/refractiveindex (V/n) map corresponding to the table is shown in FIG. 1. X- andY-channel gains were standardized using droplet suspensions of n-pentane(n=1.3577), n-hexane (n=1.3776) and n-heptane (n=1.3884). Fiftymilliliters of each hydrocarbon was vortexed with 1 ml of RBC/Basosheath reagent for 10 seconds and a sample was analyzed via directcytometry, i.e., the sample was not further diluted before it passedthrough the flowcell. A volume/refractive index map, includingtable-derived curves of constant refractive index for each of the threehydrocarbons, was displayed on the report screen, along with the actualcurves formed by the droplets. The signal gains were adjusted and thesamples were re-run, as needed, until the actual curve for eachhydrocarbon overlaid its associated table-derived curve. Examples of thepatterns that were generated are displayed in FIG. 2. Thus, inaccordance with the invention, platelets are specifically resolved fromnon-platelets by presence within a volume/refractive index map and arefurther resolved on the basis of the characteristic gaussiandistribution of platelet refractive indices.

The RBC count performable by the PLT1 method was calibrated as onH•™Systems with a TECHNICON Calibrator, in accordance with themanufacturer's instructions. The RBC count calibration factor, whichaccounts for dilution, was also applied to the platelet count (e.g., ason H•™Systems), since the platelets and the red blood cells in a sampleare subject to identical dilution factors. However, in accordance withthe invention and in contrast to the comparative automated methods, noindependent platelet-count calibration factor was applied in the PLT1system and method. Therefore, differences in the platelet count betweenPLT1 and other technologies would not be obscured by artificialcalibration factors for comparative purposes.

The PLT1 system is designed to process a whole blood sample by employingthe hydraulics, pneumatics, chemistry, reaction time, and counting timeof a RBC/PLT channel. Also, the platelet channel signals are acquiredfrom the same pair of optical detectors that are used in the RBC/PLTchannel; however, the increased signal gain amplifications acquired formeasuring and determining platelets as described above are unique to thePLT1 system and are not a part of the systems and methods presently usedin the art. The acquired signals for the system are analyzed asdescribed and exemplified hereinbelow:

Signals represent non-platelets (i.e., RBCs or "Others") if 1) they areoutside of the V/n map (these represent "Others"); 2) they saturate thedetectors (X=99, Y=99) (these represent RBCs); or 3) they are inchannels X<80, Y=99 (these represent "Others"). Signals above and to theleft of the map near the origin represent cellular debris. Largersignals, including those in channels X<80, Y=99 represent red cellghosts. Saturation signals are due to RBCs and very large platelets.Signals below and to the right of the map are also due to RBC fragments(see FIG. 3).

The remaining signals on the map are analyzed as follows: First, thesystem computes the mean refractive index and standard deviation (SD) ofsignals in channels X=18 and above, using the Mie conversion table. Thesystem excludes signals on the V/n map below channel X=18 from this partof the analysis because of possible debris contamination. Then, anyparticle signal having a refractive index value of between about +2 and-1.8 SD of the mean is designated as a platelet. The rest of theparticle signals are designated as "Others". This yields counts forthree types of particles, namely, PLTs (P), RBCs (R), and "Others" (O).The number of particles detected by the system, called V_(sig), isgreater than the number of particles analyzed. Therefore, P, R, and 0represent the relative number of each type of particle rather than theactual raw count of each type. To obtain the raw counts, the systemperforms the following conversions:

    N.sub.r =(R/(R+P+O))×V.sub.sig ; N.sub.p =(P/(R+P+O))×V.sub.sig ;

N_(o) =(O/(R+P+O))×V_(sig) ; where N_(r) =raw red blood cell count;N_(p) =raw platelet count; N_(o) =raw "other" count; and V_(sig) =totalnumber of particles detected. The raw counts are then corrected for"coincidence", i.e., the simultaneous occurrence of two or moreparticles in the detector channel, which the detector counts as a singleparticle. "Others" are assumed to behave as PLTs with respect tocoincidence and are therefore grouped with them in thecoincidence-correction calculations. The frequency of coincidence isaccurately predicted by Poisson statistics, as is appreciated by thoseskilled in the art. The corrected raw RBC, PLT, and "Others" counts aredesignated RBC', PLT' and Others'. Finally, the RBC calibration factoris applied to RBC', PLT', and Others' to yield the following values: RBC(10⁶ /μl); PLT (10³ /μl); and Others (10³ /μl).

It is envisioned that the volume and refractive index dynamic ranges ofthe improved PLT1 automated method and system can be effectivelyextended to handle the occasional appearance of large and dense PLTs inthe saturation channels X=99, Y=99. PLT1 identifies these as red bloodcells. This may be accomplished by extending the current H•™System Mieconversion table down to V<8 fl and n<1.400. The RBC/PLT Channelamplification employed in the PLT1 system provides adequate resolutionof larger platelet signals for the application of extended tables toprovide accurate V and n values.

The PLT1 system of the invention computes mean platelet volume (MPV, fl)and mean refractive index based on the Mie tables. The system alsoderives mean platelet component concentration (MPC, g/dl) and meanplatelet dry mass (MPM, pg) from the means of the refractive index andthe volume, as demonstrated below:

MPC (g/dl)=(mean refractive index--1.333)/(0.0018/(g/dl)), where1.333=refractive index of water and 0.0018/(g/dl)=average refractiveindex (RI) increment. As will be appreciated by those in the art and asdiscussed further below, the RI increment value is treated as a constantin this equation to eliminate it as a variable from cell-to-cell.

MPM (pg)=MPC (g/dl)×MPV(fl)/100. (Note that g/dl=100×pg/fl.)

The average refractive index increment value is based on the fact thatthe major components of platelet dry mass are protein (57%), lipid(19%), and carbohydrate (8.5%) (Wintrobe's Clinical Hematology, NinthEdition, 1993, page 515). The refractive index increments of thesecomponents are 0.00187/(g/dl), 0.0017/(g/dl), and 0.00143/(g/dl),respectively (S. H. Armstrong et al., 1947, J.A.C.S., 69:1747-1753; R.Barer and S. Joseph, 1954, Quarterly Journal of Microscopical Science,95:399-423). Using the relative concentrations of these components toassign an average refractive index increment to platelets yields a valueof 0.0018/(g/dl). Of the minor components, some have higher refractiveindex increments and some have lower increments. The mean increment ofthese components is not expected to significantly change the assignedvalue. Note that 0.0018/(g/dl) is also the value assigned to protoplasmby the literature in the art (R. Barer and S. Joseph, 1954 QuarterlyJournal of Microscopical Science, 95:399-423). In addition to reportingRBC and PLT counts, MPV, MPC and MPM, the system of the inventiondisplays frequency histograms of platelet volume, platelet componentconcentration, and platelet dry mass (FIG. 4).

The PLT1 system and method can use the same chemical reagents as thedoes RBC/PLT Channel method which is employed by the current TECHNICONH•™System; the sphering reagent used does not adversely affect or actupon the platelets. Moreover, additional sphering is not required forplatelets that have been collected in K₃ EDTA anticoagulant and analyzedin accordance with the invention. For example, FIG. 5A depicts a PLT1scatter/scatter cytogram generated by the PLT1 system and method of theinvention for platelets that have been anticoagulated in K₃ EDTA andthen resuspended in RBC Diluent Solution. As reported in the art, K₃EDTA spheres platelets, albeit imperfectly (S. Holme and S. Murphy,1980, J. Lab. Clin. Med., 96:480-493, G. V. R. Born, 1970, J. Physiol.,209:487-511). FIG. 5B depicts a PLT1 scatter/scatter cytogram for asecond aliquot of the same sample suspended in isotonic phosphatebuffered saline (PBS). As can be observed by comparing FIGS. 5A and 5B,the two cytograms resulting from the method and system of the inventionhave the same appearance. Moreover, the reported parameter values arealso equal, within instrumental error limits. These results demonstratethat a red blood cell sphering reagent is not required for the method ofthe invention to provide accurate RBC counts and platelet parameterresults.

In addition, FIGS. 6A and 6B show a corresponding pair of PLT1-generatedscatter/scatter cytograms assessing platelets from the same donor,except that a commercially available acid/citrate/dextrose (ACD)solution was used as the anti-coagulant. In contrast to the action of K₃EDTA, ACD is known not to sphere platelets (S. Holme and S. Murphy,1980, J. Lab. Clin. Med., 96:480-493. G. V. R. Born, 1970, J. Physiol.,209:487-511. G. V. R. Born et al., 1978, J. Physiol., 280:193-212. M.Frojmovic and R. Panjwani, 1976, Biophys. J., 16:1071-1089). Again, thecytograms have the same appearance, although samples suspended in ACDresult in cytograms that appear more diffuse than those generated forsamples suspended in K₃ EDTA. This diffuse character is due to therandom orientation of the non-spherical platelets within the flowcelland does not adversely affect the sensitivity and accuracy of theresults obtained using the system and method of the invention. Theseresults demonstrate that the red blood cell diluent does not sphereplatelets; indeed, platelet sphering is not required for the accuracy ofresults in the invention.

As described herein, the present invention demonstrates for the firsttime that sphered platelets, like red blood cells, behave "effectively"as homogeneous spheres under the chosen measurement conditions, eventhough platelets are not perfectly sphered and contain granules ofvarious types, numbers, and refractive indices. Prior to the presentinvention, this method of analysis was thought to be effective only forsphered, homogeneous particles (see, for example, U.S. Pat. No.4,735,504 to D. H. Tycko).

Another aspect of the invention is the application of Mie ScatteringTheory to two-angle scattering measurements for platelet analysis, usingthe same or similar angle intervals suitable for red blood cellanalysis. Prior to the present invention, those in the art were awarethat the selected pair of cone angle ranges used for red blood cellanalysis was specific to this cell type, since, even for red bloodcells, not all angle pairs provided accurate analyses (see U.S. Pat. No.4,735,504 to Tycko). Further, the analysis of Tycko was shown to beeffective only for sphered red blood cells, which are typically about 10times larger than platelets (i.e., MCV=85 fl for red blood cells versusMCV=8.5 fl for platelets); therefore, red blood cells provide muchlarger signals for analysis. Also, until the time of the inventiondescribed herein, those in the art were aware only that two angleintervals sufficed for the analysis of homogeneous and perfectly spheredparticles, and it was assumed that imperfectly sphered particlesrequired at least three angle intervals for analysis. The presentinvention demonstrates for the first time that the above-describedparameters (i.e., pair of cone angles and two angle intervals),previously used only for red blood cell analysis are also effective foraccurate and sensitive determinations and measurements ofimperfectly-sphered and non-homogeneous particles, such as platelets,which have normal volume ranges of about 2-20 fl.

Current automated methods (such as those of the TECHNICON H•™Systems andas described in U.S. Pat. No. 4,735,504 to Tycko) are designed only forthe analysis of red blood cells, such as for determining red blood cellvolume and cellular hemoglobin concentration. In contrast, the PLT1method of the invention advantageously allows the simultaneous analysisof platelets and red blood cells as described and exemplified herein. Ingeneral, the PLT1 method demonstrates for the first time that anautomated system, for example, the H•™System, can be configured forsimultaneous platelet and red blood cell determinations and analyses ina common optical system, in accordance with the new methodology of theinvention, without sacrificing accuracy and precision of any of thedeterminations. The separate platelet and red blood cell Mie Theoryanalyses can be performed on signals collected by a single pair ofoptical detectors in accordance with the invention, because theplatelets in a whole blood sample are analyzed under the signalamplification conditions and with the Mie Scattering Theory tables thatare particularly suitable for platelet cell type in the PLT1 system andmethod, while the red blood cells in the sample blood sample areanalyzed under amplification conditions and with Mie Scattering tablesthat are suitable for red blood cells.

Only the PLT1 method and system of the invention provide automatedmeasurements of mean platelet dry mass (MPM), whether on a cell-by-cellbasis or as a sample average. In principle, MPM (but not cell-by-celldry mass or its distribution) can be determined from measurements ofmean platelet component concentration, MPV and % platelet water, asfollows:

MPM (pg)=mean total platelet mass (pg)×(100- % water)/100, where meantotal platelet mass (pg)=mean platelet density (g/ml=pg/fl)x MPV (fl).However, these determinations require both platelet density measurements(D. G. Penington et al., 1976, Br. J. Hematol., 34:365-376; L. Corash etal., 1977, Blood, 49(1):71-85; C. B. Thompson et al., 1982, Br. J.Hematol., 50:509-519; and L. Corash et al., 1984, Blood, 64(1):185-193)and % water measurements (F. Gorstein et al., 1967, J. Lab. and Clin.Med., 70:938-950); these measurements are tedious, time-consuming, andunsuitable for high-throughput automation.

Average MPM values determined by the PLT1 system and method can becompared with indirect estimates based on established average values forplatelet density, MPV, and % platelet water. It is generally agreed inthe art that for platelets anti-coagulated in ACD and separated onstractan gradients, mean platelet density is approximately 1.065 g/ml(D. G. Penington et al., 1976, Br. J. Hematol., 34:365-376; L. Corash etal., 1977, Blood, 49(1):71-85; C. B. Thompson et al., 1982, Br. J.Hematol., 50:509-519; and L. Corash et al., 1984, Blood, 64(1):185-193)and MPV is approximately 6.5 fl (E. A. Trowbridge et al., 1985, Clin.Phys. Physiol. Meas., 6(3):221-238; L. Corash et al., 1977, Blood,49(1):71-85; C. B. Thompson et al., 1982, Br. J. Hematol., 50:509-519).However, the art-derived MPV value is based on aperture impedancemeasurements made by devices calibrated with spherical polystyrenebeads. Therefore, the MPV estimates for the non-spherical ACD plateletsare routinely low (N. B. Grover et al., 1969, Biophys. J., 9:1398; J.Hurley, 1974, Biophys. J., 10:74). All other things being equal,aperture impedance measurements on K₃ EDTA-platelets (i.e., spheredplatelets) are more accurate. These types of measurements yield MPVvalues of approximately 8.5 fl for fresh (about 1 hour old) samples (E.A. Trowbridge et al., 1985, Clin. Phys. Physiol. Meas., 6(3):221-238).

There is disagreement among those in the art as to whether or not K₃EDTA swells platelets in addition to sphering them (see, for example, S.Holme and S. Murphy, 1980, J. Lab. Clin. Med., 96:480-493; E. A.Trowbridge et al., 1985, Clin. Phys. Physiol. Meas., 6(3):221-238; G. V.R. Born, 1970, J. Physiol., 209:487-511; G. V. R. Born et al., 1978, J.Physiol., 280:193-212). It has been reported that density gradientmeasurements of K₃ EDTA platelets yielded a mean density of 1.060 g/ml(H. H. K. Watson and C. A. Ludlam, 1986, Br. J. Hematol., 62:117-124). Acomparison of this value to the value of 1.065 g/ml for ACD-plateletssuggests that K₃ EDTA swells platelets by about 8%. On this basis, theMPV for "unswelled" platelets=7.8 fl (which agrees reasonably well withpublished values based on thrombocrit measurements (S. Karpatkin and A.Charmatz, 1969, J. Clin. Invest., 48:1073-1082) and with visualmicroscopy (M. Frojmovic and R. Panjwani, 1976, Biophys. J.,16:1071-1089)). Using 7.8 to 8.5 fl as the MPV range, the mean totalplatelet mass range is 8.31 pg to 9.05 pg. Estimates of the % plateletwater content range from 74.6% to 77% (F. Gorstein et al., 1967, J. Lab.Clin. Med., 70:938-950; S. Karpatkin, "Composition of platelets", In:Hematology. 2nd Ed. 1977. McGraw-Hill, N.Y., pp. 1176-1178). This yieldsan MPM range of 1.91 pg to 2.30 pg. The mean value of 2.02 pg obtainedusing PLT1 is within this range. In contrast, the highest published MPMvalue, 2.8 pg, (S. Karpatkin, "Composition of Platelets", In:Hematology. 2nd Ed. 1977. McGraw-Hill, N.Y., pp. 1176-1178), is faroutside this range. In addition, this value is unlikely on physicalgrounds, since it equates to a component concentration range of 32.9g/dl to 35.9 g/dl, which overlaps the range of red cell componentconcentration--35 g/dl to 38 g/dl--for MCHC=32 g/dl to 35 g/dl (J. W.Harris and R. W. Kellermeyer, 1972, In The Red Cell, 2nd Ed., HarvardUniversity Press, p. 282). Accordingly, platelets of average densityshould be found within the low density fraction of normal red blood cellpopulations. However, this is not the case, as demonstrated by commonpractice.

In another aspect, measurements of platelet activity, as provided by themethod and system of the invention, are clinically useful. Currently,fluorescence flow cytometry (G. I. Johnston et al., 1987, Blood,69(5):1401-1403; J. N. George et al., 1986, J. Clin. Invest.,78:340-348); platelet density measurement (D. G. Pennington et al.,1976, Br. J. Hematol., 34:365-376; L. Corash et al. 1977, Blood,49(1):71-85; A. J. Friedhoff et al., 1978, Blood, 51(2):317-323; C. B.Thompson et al., 1982, Br. J. Hematol., 50:509-519: L. Croash et al.,1984, Blood, 64(1):185-193); and MPV determination (C. B. Thompson etal., 1983, J. Lab. Clin. Med., 101:205-213) are used to predict plateletactivity. As mentioned hereinabove, the first of these methods,fluorescence flow cytometry, is tedious, time-consuming, and expensive.The second method, platelet density measurement, is indirect as well.The third method, MPV determination, is indirect and is affected bycollection and storage conditions. Therefore, a simple, quick,inexpensive and robust method for predicting platelet activity isdesirable in the art.

Potential platelet activity increases along with the number and mass ofalpha- and dense-granules (L. Corash et al., 1977, Blood, 49(1):71-85and L. Corash et al., 1984, Blood, 64(1):185-193). Since platelet drymass correlates with granule content (Ibid.), potential plateletactivity increases with MPM. Therefore, one aspect of the PLT1 method ofthe invention provides a simple, accurate, and inexpensive method forpredicting platelet activity. Further, MPM is a robust parameter sinceit changes little in samples that have been stored for up to about 24hours prior to analysis, even at room temperature. In addition, MPMbehaves predictably over time, based on correlation coefficients (r)(see Table 1). Table 1 presents MPM and MPC determinations of normalblood donor samples versus time and temperature (8 hours versus 1 hourat room temperature (RT); 24 hours versus 1 hour at RT; 8 hours at RTversus 8 hours at 4° C.; and 24 hours at RT versus 24 hours at 4° C.)produced by the PLT1 system and method. In the relevant tables presentedhereinbelow, "r" is correlation coefficient; "syx" is standard error ofthe estimate; "Xmean" is mean value for independent variable, and"Ymean" is mean value for dependent variable. Thus, even more preciseMPM values for fresh samples can be obtained from aged or stored samplesby extrapolation, given in vitro sample age and storage temperature. Forexample, given a rate of decrease 4% per 24 hours at room temperature,and an MPM of 2.00 pg at 24 hours, the MPM of the fresh sample would be2.08 pg. It is noteworthy that the PLT1 system reports essentially thesame MPM values for ACD- and for K₃ EDTA-anticoagulated blood samples(Table 2 and FIGS. 5A and 5B). This is surprising in light of the factthat platelets are not sphered in ACD, but are sphered in K₃ EDTA.Therefore, the PLT1 method of the invention is versatile and providesaccurate and reliable MPM results for the analysis of plateletssuspended in both types of anticoagulants.

                                      TABLE 1                                     __________________________________________________________________________                              Xmean-  inter-                                      SAMPLE         r  Syx Xmean                                                                             Ymean                                                                              slope                                                                            cept                                        __________________________________________________________________________    NORMAL DONORS: MEAN PLT DRY MASS (MPM)                                        ACCURACY DATA                                                                 8 HR, RT                                                                             vs.                                                                             1 HR  0.94                                                                             0.04                                                                              2.02                                                                              0.03 0.96                                                                             0.06                                        8 HR, 4C                                                                             vs.                                                                             1 HR  0.94                                                                             0.04                                                                              2.02                                                                              0.01 0.97                                                                             0.04                                        24 HR, RT                                                                            vs.                                                                             1 HR  0.93                                                                             0.04                                                                              2.02                                                                              0.07 0.92                                                                             0.09                                        24 HR, RT                                                                            vs.                                                                             1 HR  0.93                                                                             0.05                                                                              2.02                                                                              0.03 1  -0.01                                       8 HR, RT                                                                             vs.                                                                             8 HR, 4C                                                                            0.93                                                                             0.04                                                                              2.01                                                                              0.02 0.91                                                                             0.16                                        24 HR, RT                                                                            vs.                                                                             24 HR, 4C                                                                           0.91                                                                             0.05                                                                              1.99                                                                              0.04 0.84                                                                             0.28                                        NORMAL DONORS: MEAN PLT COMPONENT CONCENTRATION (MPC)                         ACCURACY DATA                                                                 8 HR, RT                                                                             vs.                                                                             1 HR  0.61                                                                             0.73                                                                              25.6                                                                              2.8  0.74                                                                             3.97                                        8 HR, 4C                                                                             vs.                                                                             1 HR  0.45                                                                             0.62                                                                              25.6                                                                              1.7  0.41                                                                             13.4                                        24 HR, RT                                                                            vs.                                                                             1 HR  0.6                                                                              0.6 25.6                                                                              5.7  0.59                                                                             4.78                                        24 HR, RT                                                                            vs.                                                                             1 HR  0.37                                                                             1.14                                                                              25.6                                                                              3.7  0.6                                                                              6.52                                        8 HR, RT                                                                             vs.                                                                             8 HR, 4C                                                                            0.55                                                                             0.77                                                                              23.9                                                                              1.1  0.73                                                                             5.35                                        24 HR, RT                                                                            vs.                                                                             24 HR, 4C                                                                           0.42                                                                             0.68                                                                              21.9                                                                              2    0.26                                                                             14.19                                       __________________________________________________________________________

MPC is linearly related to platelet refractive index, which is in turnlinearly related to platelet density. The results of Example 4 below, inwhich MPC values obtained in accordance with the methods of theinvention were compared with fluorescence flow cytometric data for thedose response of normal platelets to the platelet agonist thrombin,demonstrate that MPC values correlate to PLT activation state (FIGS.14A-14D). Significantly, the method of the invention for studying PLTactivation is inexpensive, rapid and very simple to use. In addition,data analysis is easily automated. Moreover, the information that can beobtained by employing the method of the invention is essentially uniformfrom one instrument to another, if and when different instruments areused to carry out the method.

The method is especially suitable for the analysis of blood sampleswhich have been anticoagulated in sodium citrate or ethylene diaminetetraacetic acid (EDTA), preferably, K₃ EDTA. As appreciated by those inthe art, solutions of sodium citrate or K₃ EDTA can be mixed with ablood sample, or sodium citrate or K₃ EDTA in dry form (i.e., a powder)can be dissolved in the blood sample for use as anticoagulant. As anexemplary guide, about 7 to 14 mg of K₃ EDTA in powder form are used per7 cc tube. Sodium citrate is used routinely as a solution at 2.0 to 5g/dl, preferably, 3.2 to 3.8 g/dl per tube, and in a final ratio of 1part sodium citrate to 9 parts whole blood. As is further appreciated bythose in the art, K₃ EDTA is by far the most commonly used anticoagulantfor automated hematology analysis. Advantageously in this regard, thepresent invention provides a valuable alternative to prior methods ofmeasuring platelets and activated platelets using antibodies, whichcannot employ EDTA-containing solutions, due to the detrimental effectof such solutions on the integrity of epitopic binding sites betweenantibodies and the molecular structures to which they bind.

Another aspect of the invention is to provide a method and automatedsystem to assess the relationship between MPM values and disease statesand/or disease treatment regimens (see Example 2). The inventionprovides a convenient method of determining the effect of, for example,chemotherapy or radiation treatments on a patient's platelet granulecontent, by measuring MPM. Currently, it is believed thatthrombocytopenia associated with peripheral destruction of plateletsresults in larger-than-normal platelets, while the same condition due toreduced thrombopoiesis results in normal-sized or small platelets (J. D.Bessman et al., 1982, Am. J. Clin. Pathol., 78:150-153; R. B. Nelson andD. Kehl, 1981, Cancer, 48:954-956). Further, increased platelet size isassociated with myocardial infarction (A. Eldor et al., 1982, Br. Med.J., 285:397-400; H. A. Cameron et al., 1983, Br. Med. J., 287:449-451;J. F. Martin et al., 1983, Br. Med. J., 287:486-488). In view of thecorrelation between MPV and platelet dry mass (L. Corash et al., 1977,Blood, 49(1):71-85, and L. Corash et al., 1984, Blood, 64(1):185-193),one would expect a high MPM value to be associated with destructivethrombocytopenia and a low MPM value to be associated with reducedthrombopoiesis. In addition, high MPM is likely to be predictive ofthrombotic potential.

                  TABLE 2                                                         ______________________________________                                        PLT1 SYSTEM MPM VALUES (pg)                                                   EFFECT OF ANTI-COAGULANT                                                      NORMAL DONORS: (1 HR SAMPLES; RT)                                             DONOR#           K.sub.3 EDTA                                                                           ACD                                                 ______________________________________                                        12               1.938    1.929                                               8                1.979    2.009                                               13               2.079    2.041                                               11               2.03     2.01                                                14               1.891    1.976                                               30               1.874    1.94                                                31               1.878    1.912                                               32               2.03     2.011                                               33               2.031    2.06                                                34               1.914    1.952                                               80               2.308    2.381                                               81               1.989    2.012                                               MEAN VALUE       1.995    2.019                                               ______________________________________                                    

The following examples are illustrative of the invention. They arepresented to further facilitate an understanding of the inventiveconcepts and in no way are to be interpreted as limiting the scope ofthe present invention.

EXAMPLES Example 1

This example describes the performance of the high-gain PLT1 channel andplatelet analysis method of the invention as tested on approximately 75normal blood samples (obtained from Bayer Corporation donors). Forreference comparisons among the PLT1 method and system of the inventionand systems versus methods used in the art, the blood platelet sampleswere analyzed using the TECHNICON H•™2 automated analyzer, the CoulterSTKS analyzer, and the improved, more accurate PLT1 system. For visualreference, slides of stained blood smears were also prepared for eachsample. Platelet counts, MPV values, and RBC counts were compared amongthe analysis modes enumerated above. Data were also collected for twoparameters new to automated methodology and introduced to the art by thePLT1 system and method of the present invention, namely, Mean PlateletDry Mass (MPM) and Mean Platelet Component Concentration (MPC).

All samples were collected into evacuated containers containing K₃ EDTA.Samples were analyzed after 1 hour of room temperature storage, after 8hours both at room temperature and at 4° C., and after 24 hours at roomtemperature and at 4° C.

To perform platelet analyses using the TECHNICON H•™2 and the CoulterSTKS systems, the systems were standardized and calibrated according tothe manufacturers' instructions. All samples were run and analyzed induplicate. In addition, samples were run and analyzed in duplicate onthe PLT1 test system which was standardized and calibrated as describedhereinabove.

Film slides of blood smears were prepared in duplicate for all samples.Wright-Giemsa stain was then applied to the slides via the commerciallyavailable Hema-Tek 2000 Slide Stainer (Bayer Corporation). The slideswere stored for platelet count and relative size reference.

The results of the normal blood analysis and PLT count comparativeaccuracy data are presented in Table 3; MPV comparative accuracy dataare presented in Table 4; RBC count comparative accuracy data arepresented in Table 5; and MPM and MPC accuracy data are presented inTable 1.

                  TABLE 3                                                         ______________________________________                                        NORMAL DONORS: PLT COUNT ACCURACY DATA                                                                      Xmean-                                          SAMPLE  r       Syx    Xmean  Ymean slope intercept                           ______________________________________                                        PLT1 vs. H • ™ 2                                                     1 HR    0.97    12     265    -1    0.94  17                                  8 HR, RT                                                                              0.96    15     261    -7    0.99  9                                   8 HR, 4C                                                                              0.95    17     258    -11   1     10                                  24 HR, RT                                                                             0.95    17     252    -10   0.98  15                                  24 HR, 4C                                                                             0.94    19     250    -7    1.04  -4                                  PLT1 vs. COULTER STKS                                                         1 HR    0.98    12     275    9     0.96  2                                   8 HR, RT                                                                              0.97    15     273    5     0.98  0                                   8 HR, 4C                                                                              0.96    15     273    5     0.98  1                                   24 HR, RT                                                                             0.97    13     269    8     0.96  3                                   24 HR, 4C                                                                             0.94    19     260    3     0.96  7                                   ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        NORMAL DONORS: MPV ACCURACY DATA                                                                            Xmean-                                          SAMPLE  r       Syx    Xmean  Ymean slope intercept                           ______________________________________                                        PLT1 vs. H • ™ 2                                                     1 HR    0.6     0.29   9.1    1.1   0.36  4.76                                8 HR, RT                                                                              0.28    0.43   8.3    -0.6  0.19  7.31                                8 HR, 4C                                                                              0.46    0.43   8.5    -0    0.33  5.37                                24 HR, RT                                                                             0.43    0.43   7.2    -3    0.35  7.62                                24 HR, 4C                                                                             0.08    0.67   7.6    -1.6  -0.07 9.85                                PLT1 vs. COULTER STKS                                                         1 HR    0.8     0.22   8.5    0.5   0.41  4.51                                8 HR, RT                                                                              0.76    0.29   8.7    -0.2  0.47  4.76                                8 HR, 4C                                                                              0.88    0.23   8.8    0.3   0.58  3.46                                24 HR, RT                                                                             0.63    0.37   9.1    -1.1  0.38  6.73                                24 HR, 4C                                                                             0.85    0.36   9.4    0.1   0.67  3.02                                ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        NORMAL DONORS: RBC COUNT ACCURACY DATA                                                                    Xmean-                                            r           Syx    Xmean    Ymean slope intercept                             ______________________________________                                        PLT1 vs. H • ™ 2                                                     1 HR    0.98    0.1    4.81   0.07  0.96  0.14                                8 HR, RT                                                                              0.98    0.09   4.82   0.08  0.96  0.13                                8 HR, 4C                                                                              0.99    0.07   4.83   0.09  0.95  0.17                                24 HR, RT                                                                             0.99    0.09   4.83   0.1   0.94  0.21                                24 HR, 4C                                                                             0.98    0.09   4.83   0.09  0.94  0.22                                PLT1 vs. COULTER STKS                                                         1 HR    0.98    0.1    4.75   0.01  0.96  0.17                                8 HR, RT                                                                              0.98    0.1    4.75   0.01  0.93  0.33                                8 HR, 4C                                                                              0.98    0.09   4.75   0.01  0.94  0.28                                24 HR, RT                                                                             0.98    0.1    4.75   0.02  0.94  0.26                                24 HR, 4C                                                                             0.98    0.1    4.74   0     0.97  0.15                                ______________________________________                                    

PLT counts: The platelet counts obtained from the PLT1 method of theinvention agreed well with counts from the H•™2 and the STKS systems,both widely accepted platelet counting devices. It is noted that noplatelet count calibration factor was applied in the PLT1 method, whilethe calibration factors for H•™2 and STKS were 0.85 and 1.02,respectively. This suggests that the current H•™System technologyincludes significant numbers of non-platelets in its platelet raw count.

MPV: No current method for measuring MPV is considered to be a standardin the art. Therefore, comparisons among the methods relate to thequalitative behavior of platelets. FIG. 4 shows a typical, normal sampleplatelet volume histogram for the PLT1 method. It represents alog-normal distribution of platelet volumes, in agreement with publishedresults (J. M. Paulus, 1975, Blood, 46(3):321-336). Typical plateletvolume distributions for the H•™2 and STKS systems are also log-normal.

For normal samples, the MPV values obtained using the PLT1 system of theinvention and the comparative Coulter STKS system both indicated thatMPV increases with storage time, while the values obtained using theTECHNICON H•™2 System indicated a decrease (see Table 4). This patternfor H•™2 versus STKS and PLT1 agreed with the pattern obtained using theTECHNICON H6000™ System versus the Coulter S+System as reported byTrowbridge et al. (E. A. Trowbridge et al., 1985, Clin. Phys. Physiol,Meas., 6(3):221-2382). As reported in the Trowbridge et al. paper, theTECHNICON H6000™ System (as well as in the TECHNICON H•™2 System),platelet volume is proportional to high-angle scattering intensity (5-10degrees in H6000™ and 5-15 degrees in the H•™2 system). Mie ScatteringTheory shows that scattering into these angles is sensitive torefractive index, as explained above in the Detailed Description of theInvention. As platelets age ex vivo, they swell and become lessrefractile due to water uptake (S. Holme and S. Murphy, 1980, J. Lab.Clin. Med., 96:480-493). This swelling reduces their high-anglescattering intensity, thereby causing the H6000™ and the H•™2 systems toreport a decreased MPV when, in fact, the MPV has actually increased.However, according to measurements made by the Coulter S+System and theCoulter STKS System and by other aperture impedance devices, plateletvolume is proportional to electrical impedance. Therefore, these lattersystems correctly reported increased MPV due to swelling, sinceimpedance increases as cells swell. Because the PLT1 system of theinvention converts low- and high-angle scattering signals into volumesand refractive indices using Mie Scattering Theory, as describedhereinabove, the PLT1 system and method also correctly report theincreased MPV due to swelling.

RBC Counts: RBC counts determined by the PLT1 system of the inventionagreed well with RBC counts determined by the TECHNICON H•™2 System andthe Coulter STKS System, both of which are accepted RBC countingdevices.

MPM: FIG. 4 shows a typical and representative PLT1 platelet dry masshistogram and indicates that platelet dry mass is log-normallydistributed within a sample. This agrees with the results of electronmicroscopy studies (G. F. Bahr and E. Zeitler, 1965, Lab. Invest.,14(6):217-239) and conclusions based on density gradient measurements(L. Corash and B. Shafer, 1982, Blood, 60(1):166-171). According to PLT1system measurements, a typical MPM value for a normal sample stored for1 hour at room temperature is 2.02 pg. This value is in excellentagreement with most of the published values, which are 2.5, 2.8, 2.06,2.1 and 2.06 pg, respectively (G. F. Bahr and E. Zeitler, 1965, Lab.Invest., 14(6):217-2393; F. Gorstein et al., 1967, J. Lab. Clin. Med.,70:938-950; S. Karpatkin, 1977, "Composition of platelets", In:Hematology. 2nd Ed. McGraw-Hill, N.Y., pp. 1176-1178; T. C. Bithell,1993, "Platelets and megakaryocytes", In: Wintrobe's ClinicalHematology, 9th Ed. Vol. 1. Lea and Febiger, Philadelphia, Pa., pp.511-529; and E. E. Woodside and W. Kocholaty, 1960, Blood,16:1173-1183). The MPM value dropped only slightly over 24 hours, i.e.,by 3.5%, when samples were stored at room temperature and by 1.5% whensamples were stored at 4° C. (Table 1).

MPC: FIG. 4 also shows a typical and representative PLT1 plateletcomponent concentration histogram which is normally-distributed (i.e.,displays a normal or gaussian distribution) for fresh samples. Thisagrees with the results of density gradient measurements (H. H. K.Watson and C. A. Ludlam, 1986, Br. J. Hematol., 62:117-124; J. F. Martinet al., 1983, Br. J. Hematol., 54:337-352). The average MPC valueobtained using the PLT1 system for samples stored for 1 hour at roomtemperature was 25.6 g/dl. To compare this value to published values for% solids, it is necessary to determine the densities of the non-aqueouscomponents. As described, the relative platelet composition ofprotein/lipid/carbohydrate is 57/19/8.5 and the respective densityvalues for these components are 1.33 g/ml, 0.93 g/ml and 1.50 g/ml,respectively (R. Barer and S. Joseph, 1954, "General CytochemicalMethods", Quarterly Journal of Microscopical Science, 95:399-423); thus,the average density of solid components is 1.26 g/ml. Therefore, thevolume occupied by 25.6 g of platelet components in a dl of platelets iscalculated as follows:

25.6 g×(1ml/1.26 g)=20.3 ml or 0.203 dl. The remaining volume per dl(i.e., the water) is: 1.000 dl-0.203 dl=0.797 dl. At a density of 1g/ml, the mass of this volume of water is 79.7 g. Therefore, %solids=(25.6/(79.7+25.6))×100 =24.3%. If K₃ EDTA swells platelets by 8%,as described hereinabove, then, % solids=25.8%. This range of 24.3% to25.8% is close to the range of published values, namely, 23% to 25.4%(F. Gorstein et al., 1967, J. Lab. Clin. Med., 70:938-950; S. Karpatkin,"Composition of Platelets", In: Hematology. 2nd Ed. 1977. McGraw-Hill,N.Y., pp. 1176-1178).

MPC decreased significantly over 24 hours; it decreased by 14% forsamples stored at 4° C. and by 22% for samples stored at roomtemperature (Table 1). The statistics for MPC measured at various timesand temperatures appear in Table 6. The data show that at roomtemperature, MPC values for samples analyzed at 1, 8, and 24 hours,respectively, do not overlap with each other, within 1.5 SD. Thus, onecan determine whether a sample is 1, 8 or 24 hours old with 87%confidence by measuring its MPC. Accordingly, MPC values can be used tomonitor in vitro sample age. A variety of hematologic parameters, suchas MCV, MCHC, HCT and MPV, are sensitive to sample age. Consequently,false conclusions can be drawn regarding conditions such asmacrocytosis, hypochromia, anemia, and platelet thrombotic potential ifthe effects of sample age are ignored and if accurate and reliable dataare not obtained from aged samples. Therefore, monitoring sample age viaMPC values using the platelet analysis and counting method and system ofthe invention is expected to have significant clinical value.

Example 2

This example describes the performance of the expanded gain PLT channel(PLT1) and platelet analysis method of the invention as tested onapproximately 70 abnormal blood samples (obtained from Memorial SloanKettering Cancer Center (MSKCC), New York). All of the samples hadplatelet counts below 100,000/μl and were analyzed as described inExample 1. In addition, platelet counts were determined by phasecontrast microscopy, since the validity of the automated platelet countsis not well established at the present time for all thrombocytopenicsamples (K. Mayer et al., 1980, Am. J. Clin. Pathol., 74:135-150; P. J.Combleet and S. Kessinger, 1985, Am. J. Clin. Pathol., 83:78-80).Samples were delivered to Bayer Corporation approximately 4 hours afterthey were collected and were analyzed at room temperature upon arrival.The samples were re-analyzed after 28 hours of storage at roomtemperature. The results are presented below and in Table 7 and FIG. 12.

PLT Counts: Platelet counts obtained using the PLT1 system and method ofthe invention agreed well with the platelet counts obtained using phasecontrast microscopy for samples stored at room temperature for 4 hours.PLT1 platelet counts also agreed with TECHNICON H•™2 System counts andCoulter STKS System counts, with notable exceptions, one of which isdescribed further in Example 3 hereinbelow.

MPV: For abnormal samples, the TECHNICON H•™System and Coulter STKSSystem results agreed better with each other than with the resultsobtained using PLT1, even though the opposite was true for normalsamples. The reason for this becomes clear when representative plateletvolume histograms are compared. FIG. 8 shows that the platelet volumehistograms generated by the TECHNICON H•™2 System and the Coulter STKSSystem included interfering particles in low channels. These particlesdistorted the log-normal platelet volume distributions and caused thesystems to under-report MPV values. In contrast, the PLT1 method of theinvention excluded most of these types of interfering particles, asindicated by its log-normal platelet volume distribution. Therefore,PLT1 reported a higher MPV value than did the other systems. Inaddition, FIGS. 9A-9D show that, according to the H•™2 System and theCoulter STKS System measurements, MPV is directly related to PLT count(up to 20,000/μl), while FIGS. 10A and 10B show that according to PLT1measurements, MPV and PLT count are inversely related. Moreover,according to the literature accounts, MPV and PLT counts are inverselyrelated for normal samples and for most thrombocytopenic samples (J. D.Bessman et al., 1982, Am. J. Clin. Path., 78:150-153; J. Levin and J. D.Bessman, 1983, J. Lab. Clin. Med., 101:295-307; J. D. Bessman et al.,1981, Am. J. Clin. Path., 76:289-293; C. Giles, 1981, Br. J. Hematol.,48:31-37). Thus, the literature reports are in agreement with theresults generated by the PLT1 system of the invention. Also, microscopicrelative-size measurements on stained blood slide films indicated thatthe TCP samples contained relatively more large platelets than did thenormal samples. For normal samples, all three systems indicated that MPVand PLT counts were inversely related (FIG. 11), in agreement with thefindings reported by those in the art.

Currently, MPV is a largely ignored parameter in platelet analysis, eventhough it can be used to distinguish among various hematologic disorders(J. Zeigler et al., 1978, Blood, 51(3):479-486; M. Kraytman, 1973,Blood, 41(4):587-597; J. D. Bessman et al., 1982, Am. J. Clin. Path.,78:150-153; J. Levin and J. D. Bessman, 1983, J. Lab. Clin. Med.,101:295-307; C. Giles, 1981, Br. J. Hematol., 48:31-37. A. Eldor et al.,Br. Med. Journal, 1982, 285: 397-400. H. A. Cameron et al., Br. Med.Journal, 1983, 287: 449-451. J. F. Martin et al., Br. Med. Journal,1983, 287: 486-488. G. A. Threatte, Clin Lab Med, 1993, 13 (4):937-950). The reason for the art's disregard of MPV values is that,prior to the present invention, MPV values were considered to beunreliable. (E. A. Trowbridge et al., 1985, Clin. Phys. Physiol. Meas.,6(3):221-238; G. A. Threatte et al., 1984, Am J. Clin. Path.,81:769-772) for the following possible reasons:

1) significant differences due to blood sample storage conditions arefrequently obtained for MPV values between the conventional TECHNICONH•™System and aperture impedance instruments (E. A. Trowbridge et al.,1985, Clin. Phys. Physiol. Meas., 6(3):221-238. G. A. Threatte, 1993,Clin. Lab. Med., 13(4):937-950, U. Lippi et al., 1987, Am. J. Clin.Pathol., 87:391-393);

2) neither the conventional H•™ Systems nor the aperture impedancedevices report accurate MPVs for thrombocytopenic samples, asdemonstrated above; and 3) MPV is sensitive to collection and storageconditions (C. B. Thompson et al., 1983, Am. J. Clin. Path., 80:327-332;S. Murphy and F. H. Gardner, 1971, J. Clin Invest., 50:370-377; B. S.Full and M. B. Zucker, 1965, Proc. Soc. Exp. Biol. Med., 120:296-301; J.G. White and W. Krivit, 1967, Blood, 30(5):625-635).

In contrast, the MPV results obtained using PLT1 in accordance with thepresent invention are valid for the following reasons:

1) the qualitative and quantitative agreement between results obtainedfor normal samples stored under various conditions by independentmethods, namely, PLT1 and aperture impedance, places both PLT1 andaperture impedance MPV measurements on firmer ground; 2) PLT1 MPVresults for thrombocytopenic samples agree qualitatively with reportedliterature results under a variety of conditions; and 3) PLT1 monitorsthe effect of sample storage on MPV by measuring MPC, as discussedhereinabove.

                  TABLE 6                                                         ______________________________________                                        NORMAL DONORS: MPC STATISTICS                                                 ______________________________________                                        1 HR/RT          8 HR/RT         24 HR/RT                                     ______________________________________                                        Mean    25.60899 Mean     22.78007                                                                             Mean   19.86133                              Standard                                                                              0.062196 Standard 0.08726                                                                              Standard                                                                             0.070337                              Error            Error           Error                                        Median  25.65    Median   22.87  Median 19.825                                Mode    25.75    Mode     22.77  Mode   20.13                                 Standard                                                                              0.730638 Standard 1.025071                                                                             Standard                                                                             0.79577                               Deviation        Deviation       Deviation                                    Sample V                                                                              0.533833 Sample V 1.050772                                                                             Sample V                                                                             0.633249                              Kurtosis                                                                              0.315031 Kurtosis -0.19193                                                                             Kurtosis                                                                             0.33338                               Skewness                                                                              -0.06117 Skewness -0.25711                                                                             Skewness                                                                             0.369063                              Range   3.82     Range    5.15   Range  4.01                                  Minimum 23.8     Minimum  20.02  Minimum                                                                              18.11                                 Maximum 27.62    Maximum  25.17  Maximum                                                                              22.12                                 Sum     3534.04  Sum      3143.65                                                                              Sum    2542.25                               Count   138      Count    138    Count  128                                   Confidence                                                                            0.122989 Confi-   0.172551                                                                             Confi- 0.139184                                               dence           dence                                        ______________________________________                                                   8 HR/4C         24 HR/4C                                           ______________________________________                                                   Mean   23.91804 Mean     21.91432                                             Standard                                                                             0.061206 Standard 0.109467                                             Error           Error                                                         Median 24.03    Median   22                                                   Mode   24.03    Mode     22.58                                                Standard                                                                             0.719004 Standard 1.223883                                             Deviation       Deviation                                                     Sample V                                                                             0.516966 Sample V 1.497889                                             Kurtosis                                                                             -0.77856 Kurtosis -0.79256                                             Skewness                                                                             -0.31471 Skewness -0.16517                                             Range  3.05     Range    4.98                                                 Minimum                                                                              22.21    Minimum  19.39                                                Maximum                                                                              25.26    Maximum  24.37                                                Sum    3300.69  Sum      2739.29                                              Count  138      Count    125                                                  Confi- 0.12103  Confi-   0.216667                                             dence           dence                                              ______________________________________                                    

RBC Counts: The PLT1 system results agreed well with results obtainedfrom both the TECHNICON H•™2 and the Coulter STKS modes of analysis.

MPM: Platelet dry mass was log-normally distributed for hospitalsamples, and this distribution was recognizable even for samples thatprovided as few as 150 raw platelet signals for analysis (FIG. 8). Forhospital samples stored at room temperature for 4 hours, a typical MPMvalue was 2.04 pg, which was effectively equal to the value obtained fornormal samples stored for 1 hour at room temperature. Thus, using thePLT1 system of the invention, MPM showed the same time stability forboth hospital samples and normal samples.

FIG. 7 depicts graphs which display MPM values for normal samples undervarious time and temperature conditions (Y axis) versus MPM for samplesstored for 1 hour at room temperature (X axis). Single clusters of MPMvalues centered at 2 pg (the mean values for normals) are shown. FIG. 8displays the corresponding graph for the abnormal sample set andindicates the presence of two clusters--one centered at 2.0 pg and oneat 2.3 pg. The different clusters are likely to relate to differences inpotential thrombotic activity. The higher MPM value for hospitalsamples, i.e., abnormal samples, is indicative that the platelets inthese abnormal samples have more potential activity than those in normalsamples. As described hereinabove, the platelet analysis method of theinvention and the MPM values obtained therefrom provide a means toassess the relationship between high MPM values and disease statesand/or disease treatment regimens.

MPC: Abnormal samples stored for 4 hours at room temperature had atypical MPC value of 22.1 g/dl (Table 6), which was 0.7 g/dl less thanthe value determined for normal samples stored for 8 hours at roomtemperature. MPC values obtained using PLT1 displayed the sametime-dependence for hospital samples as for normal samples.

                                      TABLE 7                                     __________________________________________________________________________    ABNORMAL  4 HOUR OLD SAMPLES: ACCURACY DATA                                   SAMPLES (TCPS)            Xmean-                                              METHOD        r   Syx                                                                              Xmean                                                                              Ymean                                                                             slope                                                                             intcpt.                                     __________________________________________________________________________    PLT1 vs. MANUAL                                                                         PLT 0.94                                                                              7.4                                                                              35.3 0.8 0.9 2.6                                         H • ™ 2  vs.                                                                   PLT 0.93                                                                              8.2                                                                              35.3 -1.4                                                                              0.95                                                                              3.1                                         MANUAL                                                                        STKS vs. MANUAL                                                                         PLT 0.92                                                                              9.2                                                                              35.5 -2.4                                                                              1   2.2                                         PLT1 vs. H • ™ 2                                                               PLT 0.98                                                                              4.5                                                                              36.3 1.4 0.94                                                                              0.7                                         PLT1 vs. STKS                                                                           PLT 0.96                                                                              5.7                                                                              38.4 3   0.87                                                                              2.1                                         H • ™ 2 vs. STKS                                                               PLT 0.97                                                                              5.4                                                                              38.3 1.8 0.9 2.1                                         PLT1 vs. H • ™ 2                                                               MPV 0.3 1  7.2  -2.4                                                                              0.27                                                                              7.6                                         PLT1 vs. STKS                                                                           MPV 0.66                                                                              0.8                                                                              8.9  -0.7                                                                              0.54                                                                              4.8                                         H • ™ 2 vs. STKS                                                               MPV 0.59                                                                              0.9                                                                              8.8  1.6 0.51                                                                              2.7                                         PLT1 vs. H • ™ 2                                                               RBC 0.99                                                                              0.06                                                                             2.94 0.03                                                                              0.97                                                                              0.05                                        PLT1 vs. STKS                                                                           RBC 0.99                                                                              0.06                                                                             2.94 0.03                                                                              1.01                                                                              0                                           H • ™ 2 vs. STKS                                                               RBC 1   0.04                                                                             2.94 0.02                                                                              0.98                                                                              0.09                                        __________________________________________________________________________    ABNORMAL  28 HOUR OLD SAMPLES: ACCURACY DATA                                  SAMPLES (TCPS)            Xmean-                                              METHOD    PAR.(#)                                                                           r   Syx                                                                              Xmean                                                                              Ymean                                                                             slope                                                                             intcpt.                                     __________________________________________________________________________    PLTI vs. H • ™ 2                                                               PLT 0.98                                                                              4  34.3 0.9 0.97                                                                              0.2                                         PLT1 vs. STKS                                                                           PLT 0.97                                                                              4.8                                                                              35.5 2.2 0.85                                                                              3.2                                         H • ™ 2 vs. STKS                                                               PLT 0.96                                                                              5.9                                                                              35.5 1.4 0.85                                                                              3.9                                         PLT1 vs. H • ™ 2                                                               MPV 0.32                                                                              1.2                                                                              5.7  -5.6                                                                              0.47                                                                              8.6                                         PLT1 vs. STKS                                                                           MPV 0.42                                                                              1.1                                                                              9.1  -2.1                                                                              0.37                                                                              7.9                                         H • ™ 2 vs. STKS                                                               MPV 0.65                                                                              0.7                                                                              9.1  -3.4                                                                              0.4 2.1                                         PLT1 vs. H • ™ 2                                                               RBC 0.99                                                                              0.05                                                                             2.96 0.06                                                                              0.97                                                                              0.02                                        PLT1 vs. STKS                                                                           RBC 0.99                                                                              0.05                                                                             2.94 0.04                                                                              0.96                                                                              0.08                                        H • ™ 2 vs. STKS                                                               RBC 1   0.03                                                                             2.95 -0.02                                                                             0.97                                                                              0.09                                        __________________________________________________________________________

Example 3

Although most of the abnormal samples described in Example 2 producedsimilar platelet counts regardless of the measurement method used, asmall number of abnormal samples produced results that differed widelydepending upon the analysis method. One such sample from athrombocytopenic donor is described in this example. Table 8 presentsthe comparative platelet count data (samples were run in duplicate onthe automated systems) and FIG. 13 displays the histograms produced byeach of the three automated methods. As shown, only the PLT1 system ofthe invention produced platelet counts that agreed with the phasecontrast microscopy counts for both of the duplicate measurements. TheTECHNICON H•™2 platelet counts were approximately double the phasecontrast microscopy counts. The Coulter STKS System correctly reportedthe platelet count in only one of the duplicates, and blanked in theother. Also, the Coulter STKS and TECHNICON H•™2 volume histograms werenot log-normal, because they included red blood cell fragmentinterference. Therefore, these two systems provided MPV values whichwere lower than the actual value, based on microscopic observation ofthe blood smear. The volume histogram generated by the PLT1 method andsystem was log-normal, and the reported MPV was within the range forthrombocytopenic samples and was also in qualitative agreement withmicroscopic observation. Further, platelet component concentrationdetermined by the PLT1 system was roughly normally distributed andplatelet dry mass was lognormally distributed, thus demonstrating thatthe particles which were counted as platelets in the abnormal samples bythe present invention had the physical characteristics of platelets, aswell.

                  TABLE 8                                                         ______________________________________                                        ABNORMAL                                                                      THROMBOCYTOPENIC SAMPLE                                                       (DONOR #70)                                                                                        PLATELET                                                 METHOD OF            COUNT                                                    ANALYSIS             (thousands/μl)                                        ______________________________________                                        PHASE CONTRAST       58                                                       PLT1                 65/51                                                    TECHNICON H. ™2   102/90                                                   Coulter STKS         65/-                                                     ______________________________________                                    

Example 4

This example describes experiments performed to measure the doseresponse to in vitro platelet activation by thrombin. For theseexperiments, a blood sample was drawn from a normal adult human donorwho was a non-smoker and was not undergoing aspirin therapy. Sampleswere collected both in sodium citrate and in K₃ EDTA anticoagulants. Acomparison of results based on the use of anticoagulants comprising K₃EDTA with those using sodium citrate demonstrates the utility of K₃ EDTAfor platelet activation studies employing the novel methods of theinvention.

Thrombin-induced platelet activation was measured by fluorescence flowcytometric detection of an increased cell surface expression of anα-granule protein (GMP-140) using the phycoerythrin-conjugatedmonoclonal antibody, CD62-PE. The expression of GMP-140 is associatedwith α-granule release. This analysis is made possible by the inclusionof the tetrapeptide glycyl-L-prolyl-L-arginyl-L-proline (GPRP) describedby Michelson et al. (A. D. Michelson et al. 1991, Blood, 77:770-779).GPRP inhibits platelet aggregation and fibrin clotting and thereforepermits cell-by-cell analysis.

Sample Preparation:

Whole blood was diluted 1:10 in PBS supplemented to contain 0.35% bovineserum albumin. Aliquots of diluted whole blood (300 μl) were incubatedfor 5 minutes in the presence of 2.5 mM GPRP (final concentration).α-thrombin was added to the test samples at final concentrations of0.002 U/ml to 0.087 U/ml as indicated in FIGS. 14A-D. PBS was added tothe control samples. Samples were incubated for 15 minutes at roomtemperature and then were diluted 1:1 in 1% paraformaldehyde (in PBS)and incubated for 30 minutes at room temperature. Saturatingconcentrations of platelet-specific monoclonal antibodies directedagainst surface receptor GP1bIX (CD42a, FITC-conjugated and CD62-PE)were added to all samples analyzed by fluorescence flow cytometry, andthe samples were incubated for 15 minutes. This step was omitted forsamples analyzed by the method practiced in accordance with theinvention. PBS was added to a final dilution of 1:600 before the sampleswere analyzed.

Sample Analysis

Samples were analyzed by a FACScan flow cytometer using aFACStation-CellQuest acquisition and analysis software (BectonDickinson, San Jose, Calif.) equipped with an argon ion laser operatingat 488 nm. The fluorescence of FITC and phycoerythrin were detectedusing 525 nm and 575 nm band pass filters, respectively. Platelets wereidentified by their forward scatter (FSC) versus side scatter (SSC)profile, as well as by FITC-positivity (green fluorescence, FL1).Activated platelets were identified by their PE-positivity (redfluorescence, FL2), i.e., FSC vs. FL2.

After the identification of platelets by light scatter gates andFITC-positivity, the binding of activation marker CD62 was determined byanalyzing 5,000 platelet events for PE fluorescence. Background binding,measured using the isotypic antibody IgG, was subtracted from eachsample. The results were reported as mean fluorescence intensity (FI,arbitrary units) for all samples. FI is more amenable to comparison withMPC than % positivity, since neither FI nor MPC required the setting ofarbitrary thresholds. In addition, samples were analyzed by theabsorption/light scatter method of the invention within two hours ofcollection.

The method of the invention and fluorescence flow cytometry methodologywere compared by measuring platelet activation versus added thrombin(FIGS. 14A-14D). The results show that the light scattering method ofthe invention tracks thrombin dose-related platelet activation for bloodsamples anticoagulated with both sodium citrate (open circles) and K₃EDTA (open squares). As can be observed, with increasing concentrationsof thrombin, the MPC decreases.

Example 5

This example describes experiments performed to measure in vitroplatelet auto-activation in K₃ EDTA. For these experiments, a bloodspecimen was drawn from a normal adult human donor into K₃ EDTAanticoagulant. Samples were prepared and analyzed as described forExample 4, but without thrombin activation and with paraformaldehydeadded at the indicated times. Table 9 presents the results of the timecourse of platelet auto-activation in the presence of K₃ EDTA.

                  TABLE 9                                                         ______________________________________                                        Time of Sample Fixation After                                                 Collection (minutes)                                                                          FI (arbitrary units)                                                                        MPC (g/dl)                                      ______________________________________                                        5               26            26.7                                            10              29            26.4                                            15              37            26.9                                            30              87            25.2                                            45              111           24.7                                            60              142           24.4                                            ______________________________________                                    

Table 9 shows that platelets in samples stored in EDTA undergoauto-activation which increases over time. Therefore, bothfluorescence-based and light scattering-based measurements of plateletactivation must account for in vitro sample age.

Example 6

This example describes experiments performed to measure the activationstate of platelets ex vivo. For these experiments, blood specimens weredrawn from a normal human donor and from three diabetic donors. Sampleswere collected into K₃ EDTA anticoagulant. Samples were prepared asdescribed in Example 4 above, except that neither thrombin norparaformaldehyde was added. Samples were analyzed 7 hours aftercollection. The results of these analyses are shown in Table 10.

                  TABLE 10                                                        ______________________________________                                        Sample ID   FI (arbitrary units)                                                                       MPC (g/dl)                                           ______________________________________                                        Diabetic 1  426          21.1                                                 Diabetic 2  563          20.9                                                 Diabetic 3  81           24.2                                                 Normal      ND           23.2                                                 ______________________________________                                    

Table 10 indicates that both MPC and FI measurements distinguish betweenblood samples exhibiting platelet activation ex vivo (Diabetics 1 and 2)and those which do not (Diabetic 3). It can be observed that the Fl ishigher and the MPC is lower than expected, based on an examination ofFIGS. 14A-14D. This suggests that all three of the diabetic samples areactivated, but that the third sample is less activated than are thefirst two. MPC for a normal blood sample is also included to show thateven for normal samples, MPC measured 7 hours after collection into EDTAsolution is lower than for fresh samples. ND indicates not determined.The reason for the drop in MPC value and the rise in FI is thatplatelets stored in EDTA progressively auto-activate over time, as shownin Example 5 above. Nevertheless, MPC, as well as FI, distinguishbetween samples that experience in vivo platelet activation and thosethat do not.

The contents of all patent applications, issued patents, publishedarticles and references, and textbooks as cited herein are herebyincorporated by reference in their entirety.

As various changes can be made in the above compositions and methodswithout departing from the scope and spirit of the invention, it isintended that all subject matter contained in the above description,shown in the accompanying drawings, or defined in the appended claimswill be interpreted as illustrative, and not in a limiting sense.

What is claimed is:
 1. An automated method for accurately discriminatingplatelets from other cells on a cell-by-cell basis in a normal or anabnormal whole blood sample and for determining qualitative andquantitative blood platelet parameters, comprising the steps of:a)analyzing an aliquot of said sample essentially one cell at a timethrough a focused light source to produce a forward light scatteringpattern representing scattered light intensity per unit scattering angleas a function of the scattering angle, said light scattering intensitymeasured over two angle intervals, the intervals being an approximately5 to 20 degree high angle interval and an approximately 1 to 5 degreelow angle interval, in two optical channels at increased first andsecond optical channel signal values to produce two scattering intensitymeasurements sufficient to resolve platelets from non-platelets in saidsample, wherein said first optical channel signal value derives from anincrease in the gain of a high angle detector and said second opticalchannel signal value derives from an increase in the gain of a low angledetector; b) converting said first and second optical channel lightscattering signal gain values to a platelet volume value and arefractive index value of said platelets; c) converting said plateletrefractive index value to a platelet component concentration value; d)computing a platelet dry mass value as a product of said plateletcomponent concentration value and said platelet volume value of steps b)and c); e) displaying histograms of said platelet volumes, plateletcomponent concentrations, and platelet dry masses; and f) resolving saidplatelets from said non-platelets in said sample and determining saidplatelet parameters by the presence of light scatter-derived plateletsignals within a volume versus refractive index map.
 2. The methodaccording to claim 1, wherein said platelets are further resolved fromsaid non-platelets based upon a normal distribution of the refractiveindices of said platelets.
 3. The method according to claim 1, whereinthe parameters of platelet volume, platelet component concentration, andplatelet dry mass are determined, and further comprising the steps of:c)converting said first and second optical channel light scattering signalgain values to a platelet volume value and a refractive index value ofsaid platelets; d) converting said platelet refractive index value to aplatelet component concentration value; and e) computing a platelet drymass value as a product of said platelet component concentration valueand said platelet volume value of steps c) and d).
 4. The methodaccording to claim 3, wherein the parameter of platelet count isdetermined, and further comprising the steps of:f) displaying histogramsof said platelet volumes, platelet component concentrations, andplatelet dry masses; and g) determining said platelet count based onparticle volume and refractive index by placing said platelets withinsaid volume versus refractive index map.
 5. The method according toclaim 1, wherein, in step a), said increase in said gain of said highangle detector is about 8 to 15-fold and said increase in said gain ofsaid low angle detector is about 20 to 35-fold.
 6. The method accordingto claim 5, wherein, in step a), said increase in said gain of said highangle detector is about 12-fold and said increase in said gain of saidlow angle detector is about 30-fold.
 7. The method according to claim 3,wherein said platelet component concentration value of said convertingstep d) is determined by subtracting the refractive index of water fromthe computed refractive index of the particle signals and dividing theresult of said subtraction by an average refractive index increment. 8.The method according to claim 7, wherein said refractive index incrementis 0.0018 g/dl.
 9. The method according to claim 1, wherein saidplatelets are non-sphered.
 10. The method according to claim 9, whereinsaid platelets remain approximately isovolumetric.
 11. The methodaccording to claim 1, wherein red blood cell counts are obtained bycounting signals in high-gain amplification channels X=99, Y=99 as redblood cells.
 12. The method according to claim 11, wherein plateletanalysis and red blood cell analysis are performed at the same time. 13.The method according to claim 1, wherein the volume and refractive indexranges are extended to avoid error due to saturation of said first andsecond optical channels by large and dense platelets by extendingdownward the tables used under standard signal gain conditions toprovide analyses of volume and refractive index of large platelets undernormal gain conditions.
 14. The method according to claim 5 or 13,further wherein said increase in said gain of said high angle detectoris about 10-fold and said increase in said gain of said low angledetector is about 25-fold.
 15. The method according to claim 1 or claim3, wherein said two light scattering measurements serve to determine theactivation state of platelets, and wherein activated platelets have ameasurably lower refractive index and a lower component concentration(MPC) than unactivated platelets.
 16. The method according to claim 1,wherein said blood sample is anticoagulated with EDTA or sodium citrate.17. The method according to claim 15, wherein said blood sample isanticoagulated with EDTA or sodium citrate.
 18. The method according toclaim 17, wherein said blood sample is anticoagulated with EDTA orsodium citrate.
 19. The method according to claim 15, wherein saidplatelets are activated in vivo or ex vivo.
 20. The method according toclaim 15, wherein said activation state of platelets is a function ofthe in vitro age of a platelet sample.
 21. The method according to claim3, wherein said platelet component concentration value obtained in stepd) determines the in vitro age of blood samples stored at from about onehour to about twenty-four hours at room temperature or at 4° C.
 22. Themethod according to claim 3, wherein said platelet dry mass valueobtained in step e), said platelet count, and platelet granule content,or combinations thereof, determine the effects of cancer therapy orcancer treatment in a patient undergoing said therapy or treatment. 23.An automated process for accurately determining the parameters ofplatelet count, platelet volume, platelet dry mass, and plateletcomponent concentration on a cell-by-cell basis and for discriminatingplatelets from other cells in a normal or an abnormal whole bloodsample, comprising the steps of:a) analyzing an aliquot of said sampleessentially one cell at a time through a focused light source to producea forward light scattering pattern representing scattered lightintensity per unit scattering angle as a function of the scatteringangle, said light scattering intensity measured over two angleintervals, the intervals being an approximately 5 to 20 degree highangle interval and an approximately 1 to 5 degree low angle interval, intwo optical channels at increased first and second optical channelsignal gains to produce two scattering intensity measurements sufficientto resolve platelets from non-platelets in said sample, wherein saidfirst optical channel signal value derives from an increase in the gainof a high angle detector and said second optical channel signal valuederives from an increase in the gain of a low angle detector; b)converting said first and second optical channel light scattering signalgain values to a platelet volume value and a refractive index value ofsaid platelets; c) converting said platelet refractive index value to aplatelet component concentration value; d) computing a platelet dry massvalue as a product of said platelet component concentration value andsaid platelet volume value of steps b) and c); e) displaying histogramsof said platelet volumes, platelet component concentrations, andplatelet dry masses; and f) determining said platelet count based onparticle volume and refractive index by placing said platelets withinsaid volume versus refractive index map.
 24. The process according toclaim 23, wherein, in step a), said increase in said high angle detectoris about 8 to 15-fold and said increase in said low angle detector isabout 20 to 35-fold.
 25. The process according to claim 24, wherein, instep a), said increase in said high angle detector is about 12-fold andsaid increase in said low angle detector is about 30-fold.
 26. Theprocess according to claim 23, wherein said platelet componentconcentration value of said converting step c) is determined bysubtracting the refractive index of water from the computed refractiveindex of the particle signals and dividing the result of saidsubtraction by an average refractive index increment.
 27. The processaccording to claim 26, where in said refractive index increment is0.0018 g/dl.
 28. The process according to claim 23, wherein saidplatelets are non-sphered.
 29. The process according to claim 28,wherein said platelets remain approximately isovolumetric.
 30. Theprocess according to claim 23, wherein red blood cell counts areobtained by counting signals in high-gain amplification channels X=99,Y=99 as red blood cells.
 31. The process according to claim 30, whereinplatelet analysis and red blood cell analysis are performed at the sametime.
 32. The process according to claim 23, wherein the volume andrefractive index ranges are extended to avoid error due to saturation ofsaid first and second optical channels by large and dense platelets byextending downward the tables used under standard signal gain conditionsto provide analysis of volume and refractive index of large plateletsunder normal gain conditions.
 33. The process according to claim 32,further wherein said increase in said high angle detector is about10-fold and said increase in said low angle detector is about 25-fold.34. The process according to claim 23, wherein said two light scatteringmeasurements serve to determine the activation state of platelets, andwherein activated platelets have a measurably lower refractive index anda lower component concentration (MPC) than unactivated platelets. 35.The process according to claim 23, wherein said blood sample isanticoagulated with EDTA or sodium citrate.
 36. The process according toclaim 34, wherein said platelets are collected in EDTA or sodiumcitrate.
 37. The process according to claim 36, wherein said plateletsare collected in EDTA.
 38. The process according to claim 34, whereinsaid platelets are activated in vivo or ex vivo.
 39. The processaccording to claim 34, wherein said activation state of platelets is afunction of the in vitro age of a platelet sample.
 40. The processaccording to claim 23, wherein said platelet component concentrationvalue obtained in step c) determines the in vitro age of blood samplesstored at from about one hour to about twenty-four hours at roomtemperature or at 4° C.
 41. The process according to claim 23, whereinsaid platelet dry mass value obtained in step d), said platelet count,and platelet granule content, or combinations thereof, determine theeffects of cancer therapy or cancer treatment in patients undergoingsaid therapy or treatment.
 42. An automated apparatus for accuratelydetermining the parameters of platelet count, platelet volume, plateletdry mass, and platelet component concentration and for discriminatingplatelets from other cells in a normal or an abnormal whole bloodsample, comprising:a) means for analyzing an aliquot of said sampleessentially one cell at a time through a focused light source to producea forward light scattering pattern representing scattered lightintensity per unit scattering angle as a function of the scatteringangle, said light scattering intensity measured over two angleintervals, the intervals being an approximately 5 to 20 degree highangle interval and an approximately 1 to 5 degree low angle interval, intwo optical channels at expanded first and second optical channel signalgains to produce two scattering intensity measurements sufficient toresolve platelets from non-platelets in said sample, wherein said firstoptical channel signal value derives from an increase in the gain of ahigh angle detector and said second optical channel signal value derivesfrom an increase in the gain of a low angle detector; b) means forconverting said first and second optical channel light scattering signalgain values to a platelet volume value and a refractive index value ofsaid platelets; c) means for converting said platelet refractive indexvalue to a platelet component concentration value; d) means forcomputing a platelet dry mass value as a function of the product of saidplatelet component concentration value and said platelet volume value ofsteps b) and c); e) means for displaying histograms of said plateletvolumes, platelet component concentrations, and platelet dry masses; andf) means for determining said platelet count based on particle volumeand refractive index by placing said platelets within said volume versusrefractive index map.
 43. The apparatus according to claim 42, wherein,in a), said increase of said high angle detector is about 8 to 15-foldand said increase of said low angle detector is about 20 to 35-fold. 44.The apparatus according to claim 43, wherein said increase of said highangle detector is about 12-fold and said increase of said low angledetector is about 30-fold.
 45. The apparatus according to claim 42,wherein said platelet component concentration value converting meansdetermines said platelet component concentration by subtracting therefractive index of water from the computed refractive index of theparticle signals and dividing the result of said subtraction by anaverage refractive index increment.
 46. The apparatus according to claim45, wherein said refractive index increment is 0.0018 g/dl.
 47. Theapparatus according to claim 42, wherein said platelets are non-sphered.48. The apparatus according to claim 42, wherein said platelets remainapproximately isovolumetric.
 49. The apparatus according to claim 42,further comprising means for counting red blood cells at the same timeas analyzing said platelets.
 50. The apparatus according to claim 42,wherein platelet activation state can be determined based on saidplatelet component concentration.
 51. The method or process according toclaim 1 or claim 23, wherein complete red blood cell analyses andplatelet analyses are performed at the same time.
 52. The apparatusaccording to claim 42, further comprising means for performing completered blood cell analyses and platelet analyses at the same time.
 53. Anautomated apparatus for accurately discriminating platelets from othercells on a cell-by-cell basis in a normal or an abnormal whole bloodsample and for determining qualitative and quantitative blood plateletparameters, comprising:a) means for analyzing an aliquot of said sampleessentially one cell at a time through a focused light source to producea forward light scattering pattern representing scattered lightintensity per unit scattering angle as a function of the scatteringangle, said light scattering intensity measured over two angleintervals, the intervals being an approximately 5 to 20 degree highangle interval and an approximately 1 to 5 degree low angle interval, intwo optical channels at increased first and second optical channelsignal gains to produce two scattering intensity measurements sufficientto resolve platelets from non-platelets in said sample, wherein saidfirst optical channel signal value derives from an increase in the gainof a high angle detector and said second optical channel signal valuederives from an increase in the gain of a low angle detector; b) meansfor converting said first and second optical channel light scatteringsignal gain values to a platelet volume value and a refractive indexvalue of said platelets; c) means for converting said plateletrefractive index value to a platelet component concentration value; d)means for computing a platelet dry mass value as a function of theproduct of said platelet component concentration value and said plateletvolume value of step b) and c); e) means for displaying histograms ofsaid platelet volumes, platelet component concentrations, and plateletdry masses; and f) means for resolving said platelets from saidnon-platelets in said sample and determining said platelet parameters byplacement within a volume versus refractive index map.
 54. The apparatusaccording to claim 53, further comprising means for further resolvingsaid platelets from said non-platelets based upon a normal distributionof the refractive indices of said platelets.
 55. The apparatus accordingto claim 53, wherein the parameters of platelet volume, plateletcomponent concentration, and platelet dry mass are determined, andfurther comprising:c) means for converting said first and second opticalchannel light scattering signal gain values to a platelet volume valueand a refractive index value of said platelets; d) means for convertingsaid platelet refractive index value to a platelet componentconcentration value; and e) means for computing a platelet dry massvalue as a product of said platelet component concentration value andsaid platelet volume value of steps c) and d).
 56. The apparatusaccording to claim 55, wherein the parameter of platelet count isdetermined, and further comprising:f) means for displaying histograms ofsaid platelet volumes, platelet component concentrations, and plateletdry masses; and g) means for determining said platelet count based onparticle volume and refractive index by placing said platelets withinsaid volume versus refractive index map.
 57. The apparatus according toclaim 53, wherein, in a), said increase in said gain of said high angledetector is about 8 to 15-fold and said increase in said gain of saidlow angle detector is about 20 to 35-fold.
 58. The apparatus accordingto claim 57, wherein, in a), said increase in said gain of said highangle detector is about 12-fold and said increase in said gain of saidlow angle detector is about 30-fold.
 59. The apparatus according toclaim 55, wherein said platelet component concentration value determinedby said means for converting d) is obtained by subtracting therefractive index of water from the computed refractive index of theparticle signals and dividing the result of said subtraction by anaverage refractive index increment.
 60. The apparatus according to claim59, wherein said refractive index increment is 0.0018 g/dl.
 61. Theapparatus according to claim 53, wherein said platelets are non-sphered.62. The apparatus according to claim 61, wherein said platelets remainapproximately isovolumetric.
 63. The apparatus according to claim 53,further comprising means for obtaining red blood cell counts by countingsignals in high-gain amplification channels X=99, Y=99 as red bloodcells.
 64. The apparatus according to claim 63, further comprising meansfor performing platelet analysis and red blood cell analysis at the sametime.
 65. The apparatus according to claim 53, further comprising meansfor extending said volume and refractive index ranges to avoid error dueto saturation of said first and second optical channels by large anddense platelets by extending downward the tables used under standardsignal gain conditions to provide analyses of volume and refractiveindex of large platelets under normal gain conditions.
 66. The apparatusaccording to claim 65, further wherein said increase in said gain ofsaid high angle detector is about 10-fold and said increase in said gainof said low angle detector is about 25-fold.
 67. The apparatus accordingto claim 55, wherein said two light scattering measurements serve asmeans for determining the activation state of platelets, and whereinactivated platelets have a measurably lower refractive index and a lowercomponent concentration than unactivated platelets.
 68. The apparatusaccording to claim 67, wherein said platelets are collected in EDTA orsodium citrate.
 69. The apparatus according to claim 68, wherein saidplatelets are collected in EDTA.
 70. The apparatus according to claim67, wherein said platelets are activated in vivo or ex vivo.
 71. Theapparatus according to claim 67, wherein said activation state ofplatelets is a function of the in vitro age of a platelet sample. 72.The apparatus according to claim 55, wherein said platelet componentconcentration value obtained by means d) determines the in vitro age ofblood samples stored at from about one hour to about twenty-four hoursat room temperature or at 4° C.
 73. The apparatus according to claim 55,wherein said platelet dry mass value obtained by means e), said plateletcount, and platelet granule content, or combinations thereof, determinethe effects of cancer therapy or cancer treatment in patients undergoingsaid therapy or treatment.